KR20170125975A - Biomarkers for Preeclampsia - Google Patents

Abstract

The present invention relates to the use of hemopexin, free, non-cell associated fetal hemoglobin and alpha-1-microglobulin as markers for preeclampsia.

Description

Biomarkers for Preeclampsia

The present invention relates to biomarkers for preeclampsia for early onset (pre-gestational 34 weeks) pre-eclampsia and for late onset pre-eclampsia. In addition, biomarkers for prediction of fetal and maternal outcomes are identified in women suffering from preeclampsia. The biomarker is HpX in combination with i) Haemopectin (Hpx), ii) Hpx in combination with alpha-1-microglobulin (A1M), or iii) AlM and free circulating fetal hemoglobin (free HbF). The marker panel can be supplemented with other markers selected from the sum Toglobin-Fetal Hemoglobin Complex (Hp-HbF), Haptoglobin (Hp), Hemoxigenin-1 (HO-1) and Hem. CD163 and CD163 combined with Hpx may be markers for fetal birth. Both Hpx levels and activity (the latter representing Hpx-a) can be used.

Preeclampsia (PE) is associated with 3 to 8% of all pregnancies and appears clinically in the second half of ^one gestation period. The clinical features that determine PE are hypertension and proteinuria that appear 20 weeks after gestation. If PE is left untreated, it is a potentially serious condition that can cause keratin characterized by generalized seizures. The associated disease, HELLP syndrome (hemolysis, elevated liver enzymes and low platelet count) progresses more rapidly and involves maternal hemolysis. A uniform classification of the different forms of hypertension during pregnancy is important in order to be able to provide a uniform diagnosis. So far, several biomarkers have been proposed for screening in the first and second trimester, but have not yet been used in clinical practice. Furthermore, some biomarkers have been proposed to support the clinician in the diagnosis and treatment of patients.

Although the pathology of PE is not fully understood, recent studies have shown that extracellular fetal hemoglobin (HbF) is involved. Using gene expression microarray techniques and proteomics (Centlow et al. ¹⁰ ), upregulation of endothelial HbF gene and accumulation of cell-free HbF in the term PE placenta have been shown. Later, Olsson et al. ¹¹ demonstrated that women diagnosed with PE increased the plasma levels of acellular HbF and adult hemoglobin (HbA), and Anderson et al. ¹² found that serum levels of HbF and A1M in pregnant women It has been demonstrated that it has risen as fast as 10 weeks of gestation. HbF has been hypothesized to induce the production of reactive oxygen species (ROS), leading to oxidative damage to the placenta and subsequent leakage across the fetal-maternal barrier (including HbF). This hyperproduction and leakage of HbF results in an increased concentration of HbF in the maternal plasma and an additional induction of ROS and inflammation. As a result, general endothelial damage causes hypertension and proteinuria, characteristic of PE.

One of the major components of blood is protein-hemoglobin (Hb), a highly oxygen-packed oxygen-transport protein in red blood cells. Hb is a tetramer consisting of four globin subunits, each carrying a heme-group in its active center. In adults, the most common Hb isoform is HbA, a tetramer consisting of two α- and two β-subunits (α ₂ β ₂ ). In the fetus, HbF is prominent, consisting of two α-chains and two γ-chains (α ₂ γ ₂ ). Furthermore, heme consists of an organo-cyclic protoporphyrin IX containing a ferrous (Fe ²⁺ ) iron atom with high affinity for free oxygen (O ₂ ). The ferrous Hb bonded to O ₂ represents oxy Hb. Autoxidation of oxy Hb is a spontaneous oxidation-reduction reaction that ultimately leads to the production of various ROS including ferric (Fe ³⁺ ) Hb (metHb), peryl (Fe ⁴⁺ ) Hb, free heme, to be. These compounds are chemically very reactive and have the potential to induce tissue damage and cell destruction by a one-electron reaction with biomolecules.

Hb is normally found to be surrounded by red blood cell membranes. The autoxidation of intracellular oxy Hb and the formation of free radicals downstream are largely prevented by superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). However, under healthy conditions and enormous amounts, a significant amount of Hb escape from red blood cells may be released during a pathological condition involving hemolysis. Thus, a number of defense mechanisms evolve both in plasma and outside the blood vessels to counter the chemical threat of acellular Hb on exposed tissue.

Haptoglobin (Hp) is probably the best studied Hb-clearance system. This binds to plasma-free acellular Hb ^19,20 , binding to the macrophage receptor CD163 ²¹ clears the Hp-Hb complex from the blood. The Hp molecule consists of two chains (? And?), And there are two allelic variants (? 1 and? 2) of the? -Chain. As a result, three phenotype variants (Hp 1-1, 1-2 and 2-2) occur in the human population. Blood free heme is isolated by hemo peksin (Hpx), Hpx- heme complex is the clearance from the circulation by the liver cell receptor CD91 ^25. In intracellular compartments, heme oxygenase (HO) is the most essential heme phasing protein that converts heme into free iron, biliverdine and carbon monoxide (CO). Plasma- and extracellular protein alpha-1-microglobulin (A1M) binds, degrades heme, and decreases metHb. A1M also acts as an antioxidant by reducing the radicals produced by downstream ROS and acellular Hb and other sources and by covalent bonding.

Increased synthesis and accumulation of acellular HbF appeared in the PE placenta. Furthermore, increased concentrations of HbF appear in maternal plasma / serum in early and late pregnancy with complications by PE, suggesting that this is an important factor linking stage 1 and 2 in pathology. Free HbF has been shown to cause placental tissue damage and oxidative stress, resulting in leakage through the blood-placental barrier into the maternal circulation. To prevent toxicity of Hb and its degraded metabolite heme and free iron, some protective scavenger systems protect the human body. Hp is the best-described Hb scavenging system that binds free Hb and transports it to macrophages and hepatocytes when its absorption is facilitated by CD 163 receptor-mediated inclusion. Within the cell, the compartment of mainly macrophage Hb is cleaved to heme by lysosomes, and heme is further cleaved by HO-1 to biliverdine, CO and free ions. Then, biliverdine is reduced to bilirubin, which is excreted through the bile system. CO has a dilating effect on the blood vessel as it relaxes the smooth muscle layer of the blood vessel and consequently lowers blood pressure.

Hpx is a circulating plasma glycoprotein that is mainly synthesized in the liver. It acts as an acute phase reactant and binds to the free heme with high affinity. The heme affinity for Hpx is influenced by several factors such as reduced pH, reduced state of heme iron atom, binding of nitric oxide (NO) to heme iron atom or presence of chloride anion and bivalent metal ion. Sodium cations increase the heme affinity for Hpx. The Hpx-heme complex is transported into macrophages and hepatocytes to express the LDL receptor-related protein 1 (LRP1, also known as CD91), which facilitates absorption of the Hpx-heme complex. Hpx has indeed been shown to prevent endothelial damage in mouse models. In addition to heme-binding, Hpx also has other activities in plasma (Hpx activity). This includes enzymatic serine protease activity, inhibition of cell adhesion, attenuation of inflammation and downregulation of angiotensin II receptors in monocytes, endothelial cells and rat aortic rings.

Based on the results provided in the experiments reported herein, the present invention provides:

i) For both early and late onset preeclampsia, markers for preeclampsia include hemopexin and alpha-1-microglobulin,

ii) For both early and late onset preeclampsia, markers for preeclampsia include hemopexin, alpha-1-microglobulin and glass, non-cell-associated fetal hemoglobin

iii) As both early and late onset preeclampsia, markers for preeclampsia include hemopexin or A1M,

iv) As a marker for preeclampsia for late onset preeclampsia,

v) As markers for preeclampsia, haptoglobin-fetal hemoglobin complex,

vi) As markers for preeclampsia, hemopexin and HO-1 (hemoxigenase),

vii) As markers for preeclampsia, a combination of hemopexin, haptoglobin, free fetal hemoglobin and hemoxigenase,

viii) As markers for preeclampsia, a combination of hemopexcin, haptoglobin, free fetal hemoglobin, hemoxigenase and alpha-1-microglobulin,

ix) Use of a combination of hemopexin and haptoglobin as markers for preeclampsia

x) The use of a combination of hemopexin, haptoglobin and haptoglobin-fetal hemoglobin as markers for preeclampsia,

xi) The use of a combination of hemopexin and hemoxigenase as markers for preeclampsia,

xii) The use of a combination of hemopexin, haptoglobin, free fetal hemoglobin and hemoxigenase as markers for preeclampsia,

xiii) The use of a combination of hemopexin, haptoglobin, free fetal hemoglobin, hemoxigenase and alpha-1-microglobulin as markers for preeclampsia,

xiv) The use of a combination of hemopexin-activity, hemopexin level, haptoglobin, free fetal hemoglobin, hemoxigenase and alpha-1-microglobulin as markers for preeclampsia,

xv) The use of either i) to v) together with free circulating fetal hemoglobin as markers for preeclampsia and / or with alpha-1-microglobulin,

xvi) A method for diagnosing preeclampsia, early preeclampsia or late onset preeclampsia,

xvii) A method for evaluating progression or regression of preeclampsia, and

xviii) A method for assessing the efficacy of preeclampsia therapy.

xix) A) fetal hemoglobin and / or b) any use of any of the above with alpha-1-microglobulin for evaluating the effectiveness of treatment of preeclampsia;

In any of the above-mentioned situations, heme may also be included.

The combination of the markers and markers mentioned above may be used as a predictive, prognostic and / or diagnostic marker.

The present invention also provides a predictive biomarker for a variety of maternal and fetal outcomes:

i) As a predictive marker for predicting tolerance to the Neonatal Intensive Care Unit (NICU), free fetal hemoglobin and / or haptoglobin and / or hemopexin

ii) Hemopexin as a predictive marker for prematurity.

iii) Use of any of i) to ii) with alpha-1-microglobulin as a predictive marker for predicting admission or premature birth to NICU.

Justice

In this specification, "singular" means "one or more" unless specified otherwise.

Hemoglobin A (HbA). There are several types of Hb. The adult Hb (HbA) consists of two alpha and two beta polypeptide chains (Hb ?, Hb?), Each consisting of a non-peptide heme group that reversibly binds to a single oxygen molecule. Hb A2, another adult Hb component, is composed of two alpha chains and two delta chains (Hb ?, Hb?).

Fetal hemoglobin (HbF). HbF, a fetal hemoglobin, consists of two alpha chains and two gamma chains. The term "fetal Hb" refers to any subunit of free HbF or HbF and includes a HbF independent in the form of a polypeptide (protein) or a nucleotide (RNA), except when applied as a target for therapy. "HbF "," fHbF "or" free HbF "refer to glass fetal hemoglobin as shown below.

As used herein, the term "free Hb" refers generally to free Hb and refers to any free Hb, free HbA, free HbA2, free HbF, any free Hb subunits (e.g. Hb ?, Hb ?, Hb? Or any combination thereof. Which additionally comprises these Hb integrants in one of the polypeptide (protein) or nucleotide (RNA) forms, except when applied as a target for treatment. The term "glass" refers to any Hb that is in the circulating liquid phase (e.g., plasma and serum, etc.), i.e., outside the red blood cells but not the red blood cells, Include; An example of a protein-linked Hb is Hp bound to Hp or Hpx. In addition, the term also includes Hb contained in STMB. Generally, the term encompasses Hb that is not contained in intact erythrocytes. Thus, the term "free Hb" encompasses all forms of Hb not contained in intact RBCs.

As used herein, the term "glass" as used in the expressions "free Hb "," free fetal Hb ", or "free Hb subunit (e.g., Hbα, Hbβ, Hbδ or Hbγ chain) On the contrary, it refers to Hb, fetal Hb or Hb subunit freely circulating in a biological fluid, which refers to the molecule remaining in the inner cell. In this sense, the term "glass" is thus primarily used to distinguish between glass Hb and Hb present in intact erythrocytes. The term does not exclude Hb contained in the STMB, but excludes Hb which, for example, binds to a protein but is still present outside the red blood cell. The present disclosure relates to free HbF and is used herein to differentiate free HbF from HbF and the same notation applies to HbF present in uninfected red blood cells. The term does not exclude HbF contained in the STMB, but excludes, for example, HbF that is bound to a protein but is still present outside the red blood cell.

As used herein, the term " marker "or" biomarker "refers to a biomolecule, preferably a polypeptide or protein, that is differentially present in a sample taken from a pregnant mammal, preferably a female.

The term "biomarker panel" is used herein for a combination of two or more biomarkers that must be measured to obtain reliable and reproducible results. Thus, the biomarker panel for predicting, diagnosing or evaluating the risk for PE progression can be a combination of Hpx and A1M, or a combination of Hpx, A1M and free HbF. It is envisaged that additional biomarkers that may be included in such a biomarker panel should be selected from the group consisting of Hp-HbF, Hp, HO-1 and heme.

The term "biological sample from a pregnant female mammal ", the term" subject "or synonym thereof is intended to mean a sample from the maternal side itself; Thus, the sample is not obtained, for example, from fetus or amniotic fluid. The term "sample from fetal or fetal placental circulation" refers to a sample taken from a fetus, e.g., from a fetal circulatory system, including blood vessels in the umbilical cord and placenta.

As used herein, fetal Hb, abbreviated as HbF, refers to the type of Hb that is the predominant component of Hb in the fetus. Fetal Hb has two alpha and two gamma polypeptide chains (Hbα, Hbγ). In the present context, HbF is a free circulating HbF, i.e. it is extracellular, but it can be bound to another substance, such as a protein that binds to Hp, even though it is not bound to other proteins.

Alpha-1-microglobulin (A1M) as used herein refers to a member of the lipocalin family referred to as alpha-1-microglobulin. Alpha-1-microglobulin can be referred to in the literature as A1M, alpha ₁ m, HI30, protein HC and alpha-1-microglycoprotein.

In the following, different methods of the present invention are discussed. In the individual phrases, a number of different detailed descriptions are given, for example regarding the characteristics of the sample, the reference or control value, the sampling time, the desired marker panel, and the like. The disclosure provided under one heading is also appropriate for the other heading, but is not necessarily repeated. Under some titles, this means that it is clear that even though there is no mention of the characteristics of the sample, for example, the subject covered under the diagnostic aspect also applies to the situations mentioned under another aspect.

Methods for diagnosing biomarker (s) and preeclampsia for preeclampsia

According to the present invention, there is provided a method for diagnosing PE and for assisting diagnosis, comprising determining whether a pregnant female has or does not have PE, or whether or not PE is in an increased risk to progress, Is provided:

(a) obtaining a biological sample (e.g., blood, plasma, urine, cerebrospinal fluid (CSF), placental biopsy (CVS), uterine fluid and / or a sample from amniotic fluid and saliva) from a pregnant woman;

(b) measuring levels of one or more biomarkers selected from Hp-HbF, Hp, Hpx, HO-1 and biomarker (s) free HbF and / or A1M levels; or

Measuring levels of Hpx and HO-1;

or

The level of one or more biomarkers selected from Hp-HbF, Hp and the levels and Hpx-activity of the biomarker (s) selected from i) free HbF and / or A1M and / or ii) Hpx and HO- step;

And

(c) comparing the measured biomarker (s) level in the sample to a reference value.

More particularly, the present invention relates to a method for the diagnosis or diagnosis of a PE comprising the following steps to determine whether the pregnant female has or does not have PE, or whether or not the PE is in an increased risk to proceed The method provides:

(a) obtaining a biological sample (e.g., blood, plasma, serum, urine, cerebrospinal fluid (CSF), placental biopsy (CVS), uterine fluid and / or a sample from amniotic fluid and saliva) from a pregnant woman;

(b) measuring the level of one or more of i) Hpx, ii) Hpx and A1M, or iii) Hpx, A1M and free HbF and optionally Hp, HO-1 and Hp-

(c) Comparing the measured biomarker (s) level in the sample to a reference value.

In some cases, (b) may also be extended to include A1M and optionally one or more of Hp, HO-1 and Hp-HbF (i.e., no use of Hpx or free HbF).

As mentioned above, preferred marker panels for use in predicting, diagnosing or assessing the risk for PE progression according to the present invention are selected by one or more of the following free HbF, Hp-HbF, Hp, HO-1 Lt; RTI ID = 0.0 > A1M < / RTI >

Control data or reference values are obtained by measuring the marker levels mentioned above in pregnant women with no progression of PE. Since the level of individual markers may vary according to gestational age, the control or reference value is preferably obtained from a pregnant woman with the same gestational age (± 1 week). As can be seen from FIG. 11 of this specification, the normal level - as well as the level at which the PE is likely to progress - varies depending on the pregnancy command the sample is taken from. However, even when this data on the reference value is not available, the difference between the control level and the risk level increases with time, and thus even when comparing the test sample taken at 20 weeks with the reference value taken at 15 weeks, It should be clear from Fig.

In the method referred to herein, it is clear that the reference value refers to the actual marker in question.

Samples can be taken at any gestational age. A given example indicates that the sample can be taken at 6 to 20 weeks or at 34 to 40 weeks of gestation. The advantage of having a panel of markers or markers that are already believed to be in short pregnancy is that early risk of PE progression can be predicted and prophylactic treatment can be initiated to reduce or avoid PE symptoms. In addition, as can be seen from the examples, the marker panel of the present invention can enable evaluation of whether the initial or late start of the PE is expected.

The biological sample is preferably a blood sample, such as a plasma or serum sample, because such a sample is most readily available.

The present invention also collects data to assist in predicting, diagnosing and evaluating the risk that the PE will progress, or data to assist in assessing the specific therapeutic or prophylactic treatment of the PE.

If desired, the sample may be prepared to enhance the detectability of one or more of the biomarkers. Typically, sample preparation involves fractionation of the sample and collection of the fraction determined to contain the biomarker (s). Pre-fractionation methods include, for example, centrifugation, RNA / DNA extraction, size exclusion chromatography, ion exchange chromatography, gel electrophoresis, liquid chromatography, protein fragmentation and protein denaturation.

The step of measuring the level of the biomarker (s) may include, for example, immunological assays (e.g., ELISA or other solid phase-based immunoassays such as SPRIA or amplification ELISA, so-called IMRAMP) PCR amplification, surface enhancement laser desorption / ionization (SELDI), high performance liquid chromatography, mass spectrometry, in situ hybridization, immunohistochemistry, chemiluminescence, tribo analysis / turbometry, lateral flow or pure or polarized fluorescence It may involve electrophoresis. However, it will be apparent to those skilled in the art that such an enumeration of techniques is not complete and that these techniques are not the only suitable methods that can be used in the present invention to measure biomarker (s) levels.

The HbF under detection and / or measurement in the method of the present invention includes any Hb chain (Hb ?, Hb ?, Hb? And Hb?), Or any combination thereof. The gamma chain represents HbF, for example, the beta and delta chains represent HbA. Based on the teachings of the present disclosure, those skilled in the art will know the Hb chain (s) to be measured. The Hp molecule consists of two chains, namely, alpha and beta, and there are two allelic variants (alpha 1 and alpha 2) of the alpha chain. As a result, mutants of three phenotypes are generated in the human populations Hp1-1, Hp1-2 and Hp2-2. The term Hp includes all these variants. A1M is present in free form and in the form of complexes bound to other proteins, such as IgA, albumin, prothrombin, and the like, and small molecules and substances (including, for example, heme or radical). Based on the teachings of the present disclosure, one of ordinary skill in the art will be aware of the A1M form (s) that should be measured.

Immunological assays (immunoassays) can be used to measure biomarker levels in accordance with the present invention. Immunoassays are assays that use antibodies that specifically bind to an antigen (e. G., Hpx). Immunoassays are characterized by the use of specific binding characteristics of specific antibodies to isolate, target and / or quantify the antigen. Thus, under the indicated immunoassay conditions, the specific antibody binds to a specific protein at least twice the background, and does not substantially bind to a significant amount for other proteins present in the sample. Using purified markers or their nucleic acid sequences, antibodies that specifically bind to a marker (e.g., Hpx) can be prepared using any suitable method known in the art (see, for example, Coligan ³⁵ ].

The biomarker level (s) can be measured, for example, using an immunological assay. In particular, immunological assays are ELISA. However, as described above, other immunological principles may also be used.

Free HbF (or other biomarker) levels can be determined by measuring free HbF (or other biomarker) RNA. In particular, free HbF messenger RNA (mRNA) is measured using real-time PCR. If total Hb levels are also determined, this level can also be determined by measuring, for example, Hb alpha chain mRNA by using real-time PCR.

Generally, a sample obtained from a subject can be contacted with an antibody that specifically binds to the marker. Alternatively, the antibody can be immobilized on a solid support (but not excluding other non-solid supports) to facilitate washing and subsequent isolation of the complex prior to contacting the antibody with the sample. Examples of solid supports include, for example, glass or plastic in the form of micro-quantum plates, sticks, beads or micro-beads.

After incubating the sample with the antibody, the mixture can be washed and the formed antibody-marker complexes detected. This can be accomplished by incubating the wash mixture with a detection reagent. The detection reagent may be, for example, a second antibody labeled with a detectable label. Exemplary detectable labels include magnetic beads, fluorescent dyes, radioactive labels, amplification kits with enzymes and thiamide (e. G., Mustard radish peroxidase, alkaline phosphatase and others commonly used in ELISA), and calorimeter markers Such as colloidal gold or colored glass or plastic beads.

Alternatively, the markers in the sample may be analyzed indirectly (e. G., Second, the labeled antibody is used to detect the bound marker specific antibody) and / or the competitive or inhibition assay (e. G., A separate epitope of the marker ≪ / RTI > is incubated concurrently with the mixture).

The method for measuring the amount or presence of the antibody-marker complex can be performed by, for example, detecting fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, refractive index (for example, surface plasmon resonance, A mirror method, a gating coupler waveguide method, or an interference measurement) or radioactivity. Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Electrochemical methods include voltage and current methods and amperometry methods. Radio frequency methods include multipole resonance spectroscopy.

Useful assays include, for example, enzyme immunoassays (EIA) such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassays such as RIA and SPRIA; Western blot analysis; Or slot blot analysis, but do not exclude other formats identified by those skilled in the art.

The step of measuring the biomarker (s) level may also be accomplished by detecting and measuring the free mRNA cipher for the Hb polypeptide in the sample, for example by detecting the mRNA sequence cipher for the biomarker or fragment thereof in the body fluids mentioned .

In the step of comparing the biomarker level in the sample with the reference value, the term "reference value" in the context of the present invention refers to the average level of the determined reference or biomarker, i.e., the same classification of the biomarker being measured in step (b) Quot; Preferably, the control comprises PE or any other pregnancy related complications, such as pregnant female mammals (preferably female) diagnosed with or suffering from pregnancy related hypertension.

It is important that the control sample (s) used or the control value (s) used are representative for the current patient sample analyzed as the normal level of the biomarker is changed by the gestational age of the pregnant woman. Controls are samples taken from pregnant women who are not at risk for progressive PE or are at risk of progression, and are sampled at similar pregnancy orders (ie, same number of pregnancy weeks). In addition, the value may depend on the analysis being applied. Thus, if an ELISA assay is used, a different value can be obtained compared to, for example, the value obtained when the radioimmunoassay is applied. Those skilled in the art will find that there is no difficulty in selecting the same assay method for the test and control samples and will have no difficulty knowing how to identify the correct gestational age for test and control samples.

As can be seen from the examples herein, reliable and reproducible results are obtained when taking samples at 34-40 weeks gestational age and when taking samples at 6-30 weeks gestational age. Most importantly, therefore, the present invention provides a reliable and biomarker (s) that can be used very early in the pregnancy to predict and / or diagnose the risk of PE progressing. As can be seen from Study II reported herein, reliable and reproducible results are obtained when the sample is taken at 6 to 20 weeks, significantly 12 to 14 weeks of gestation. In particular, the results for A1M, Hpx and HbF depend on the results reported in Study I, taking samples at 34-40 weeks gestation. Thus, the present invention provides a reliable marker for PE by 12 weeks (or earlier) and at birth.

As an example, the following values are taken as normal values when taking a sample at 34-40 weeks gestational age and when using an assay to test Hp, Hpx is an ELISA, and the assay used to test the non- Radioimmunoassay. The value given below relative to the steady state value given for A1M in the prior patent application WO 2011116958 A1 is higher because the value given in WO 2011116958 A1 is incorrect but the value reported in A1 M and WO 2011116958 A1 in WO 2011116958 A1 are two separate And that it is derived from the sample obtained at different pregnancy orders. ≪ RTI ID = 0.0 > [0040] < / RTI > This illustrates the importance of using the same method to get accurate conclusions. For HbF, there may also be a difference from the value found in WO 2008098734. With respect to Study I described below, only the non-protein-bound HbF was measured, whereas in Study II and WO 2008098734, the total HbF (i. E., Including the protein-binding moiety) was measured.

When using a control, the baseline value of the biomarker is performed using standard assay methods well known in the art. The value will, of course, vary depending on, for example, the type of assay used, the type of biomarker being measured, and the group of subjects. For example, normal average plasma levels of pregnant women not diagnosed with PE and measured by ELISA or radioimmunoassay as described above

i) ranging from 2 to 5 ng / ml (median level 3.85 ng / ml) (4.2 to 7.4, median 5.6 g / ml) for the free non-protein bound HbF,

ii) a range of 0.003 to 1.18 g / ml (median level 0.59 g / ml) for Hp-HbF,

iii) a range of 1.04 to 1.30 mg / ml (median level 1.17 mg / ml) (0.915 to 1.028, median 0.971 mg / ml) for Hp

iv) a range of 0.88 to 0.98 mg / ml (median level 0.93 mg / ml) (1.111 to 1.175, median 1.143 mg / ml) for Hpx

v) a range of 27.89 to 31.97 g / ml (median level 29.93 g / ml) (14.9 to 16.1, median 15.5 g / ml)

vi) in the range of 4.69 to 5.9 ng / ml (median level 5.29 ng / ml) for HO-1,

vii) from 52.34 to 67.38 g / ml (median level 59.86 g / ml) against heme,

Control samples are taken at 34-40 weeks gestation age (when the sample is taken at 12-14 weeks gestation age, the corresponding value is given as the insertion phrase).

However, as noted above, other steady-state values are expected when a sample is taken at another gestational age. Thus, it is desirable to use a pregnancy-corrected control value when compared to a value obtained from a test sample having a "normal" value.

As exemplified in Study II of the present specification and using the analysis described in this study, the Hpx level in plasma / serum samples from pregnant women who were taken in 6- to 20-week gestational age is less than 1.1 mg / If the cell HbF level is above 5.6 g / ml and the A1M level is above 15.5 g / ml, the pregnant woman is at increased risk of progression or progression of PE. When the median is used, the pregnant women should have a Hpx level of 1.06 mg / ml or less, a cell-free HbF level of 10.8 占 퐂 / ml or higher in the plasma / serum sample from a pregnant woman who has been taken at 6 to 20 weeks gestational age, Ug / ml, PE is in progress or is at increased risk to progress.

Higher levels of Hp-HbF, HbF, heme and / or A1M in the sample relative to the reference value are obtained when the reference (or normal) value is the level of Hp-HbF, HbF, Indicating that the pregnant woman has PE or is at increased risk for progression of PE.

The lower value of Hp, HO-1 and / or Hpx in the sample relative to the reference value, if the reference value is the Hp, HO-1 and / or Hpx level in the sample from the control group, Indicating that PE is at an increased risk to proceed.

As can be seen from the results of the present specification, the marker can also be used to determine the risk that an early or late onset PE will progress. In particular, marker Hpx activity and / or free HbF are considered herein to be important. Thus, the combination of the higher free HbF concentration and the lower Hpx activity in the sample compared to the control sample shows the late initiation, while the combination of the unchanged Hpx activity and the higher free HbF concentration results in an initial Start PE (see Table 3).

Frequent measurements of the level of one or more biomarkers may then be followed from the same type of biological sample of the same pregnant woman following the progression (or regression) of the disease.

Another way to determine the correct plasma level of a biomarker to determine whether a pregnant woman is at risk for PE or whether it already has indications is to look at the standard deviation of the tests performed when determining plasma / serum levels. Suitable parameters are assumed herein to be increased / decreased (e.g., in plasma) above the normal level by a factor of 1.1 above the standard deviation. Alternatively, the change in level should be at least 5% above the normal value.

The present invention also contemplates the use of the methods described herein in combination with other diagnostic methods. Diagnostic methods that may be used in combination with the methods of the present invention include current methods of diagnosing or aiding diagnosis of preeclampsia known to those skilled in the medical arts, examples of which are described herein before. Biological samples may be initially analyzed by the methods described herein. The biological sample can then be tested by other methods to validate the observation. Thus, the accuracy of the diagnostic method of the present invention can be improved by combining it with other diagnostic methods.

As mentioned previously, all the details mentioned under the diagnosis aspect also apply to the method described in the other aspect.

Assessment of progression / degeneration of preeclampsia

In a further embodiment of the invention, the biomarker may be used to determine the PE status (e.g., progress or regression). Some biomarkers may be used to predict a patient's prognosis, i. E., The outcome of a disease. For example, the concentrations of HbF, Hp and / or Hpx correlate with the need for clinical outcomes, such as NICU treatment, preterm birth, and cesarean section, even if other clinical indications are excluded.

Thus, in accordance with the present invention, there is provided a method of monitoring progression or regression of preeclampsia comprising the steps of:

(a) At least one biomarker selected from Hp-HbF, Hp, Hpx, HO-1 in a first biological sample isolated from a pregnant female mammal such as blood, plasma / serum, urine, CSF, placental biopsy, uterine fluid or amniotic fluid And the level of the biomarker (s) of free HbF and / or A1M,

Measure Hpx and HO-1 levels; or

Measuring at least one biomarker level selected from Hp-HbF, Hp and i) free HbF and / or A1M and / or ii) biomarker (s) level selected from Hpx and HO-1; or

(b) Measuring the same marker level selected under (a) in a second biological sample as referred to herein, isolated from said pregnant female mammal at a later time; And

(c) With the values measured in steps (a) and (b)

i) an increase in HbF, Hp-HbF, and / or A1M level (s) in the second sample relative to HbF, Hp-HbF, and / or A1M level (s) in the first sample, and /

ii) the reduction of the Hp HO-1 and / or Hpx level (s) in the second sample relative to the Hp, HO-1 and / or Hpx level (s)

Indicates the progress of the PE progress; The reduction of i) and / or the increase of ii) described above is indicative of PE degeneration.

More specifically, the present invention provides a method of monitoring progression or regression of a PE comprising the steps of:

(a) I) Hpx, ii) Hpx and A1M, and / or iii) Hpx levels in a first biological sample isolated from a pregnant female mammal such as blood, plasma, urine, CSF, placental biopsy, uterine fluid or amniotic fluid, Measuring the level of at least one of Hp-HbF, Hp, HO-1,

(b) Measuring the level of the same marker selected under (a), in a second biological sample as referred to herein, isolated from said pregnant female mammal at a later time; And

(c) Comparing the measured values in steps (a) and (b)

i) an increase in free HbF and / or A1M level (s) if measuring Hp-HbF in the second sample relative to the level in the first sample, and / or

ii) if the Hp and / or HO-1 level (s) in the second sample is measured relative to the level in the first sample,

PE progress; The reduction of i) and / or the increase of ii) described above is indicative of PE degeneration.

In some cases, (a) may also be extended to include A1M and optionally one or more of Hp, HO-1 and Hp-HbF (i.e., no use of Hpx or free HbF).

As mentioned above, preferred marker panels for use in predicting, diagnosing or evaluating the risk of progression of PE and according to the present invention are Hpx and A1M optionally supplemented with one or more of the following: glass HbF, Hp-HbF, Hp, HO-1.

An increase in HbF, Hp-HbF, heme and / or A1M level (s) or a decrease in Hp, HO-1 and / or Hpx level (s) corresponding to a standard deviation of 1.1 or greater is indicative of progression of preeclampsia and / It is assumed to represent an increased risk. Alternatively, a 5% variance from the steady state value is considered an increase (or decrease if appropriate). In a similar manner, a decrease in HbF, Hp-HbF, heme and / or A1M level (s) corresponding to a standard deviation of at least 1.1 (or 5% deviation as mentioned above) or a decrease in Hp, HO-1 and / or Hpx levels (S) is indicative of a reduced risk for PE and / or disease regression.

The detailed description referred to in the first aspect also applies to this and to the following aspects.

Methods for assessing the efficacy of certain treatments for preeclampsia

The biomarker (s) and method described above may also be used to assess the therapeutic efficacy of PE. The only difference is that the first sample is taken before treatment (indicating t ₀ ) or during treatment (indicating t ₁ ), while the second sample is taken at any time after t ₀ or t ₁ at the appropriate time. The method includes the steps of:

(a) the level of one or more biomarkers selected from Hp-HbF, Hp, Hpx, HO-1 in a first biological sample isolated from a pregnant female mammal, e.g. blood, plasma or urine, And the level of the biomarker (s) of free HbF and / or A1M; or

Measuring levels of Hpx and HO-1; or

Measuring at least one biomarker level selected from Hp-HbF, Hp and i) free HbF and / or A1M and / or ii) biomarker (s) level selected from Hpx and HO-1;

(b) measuring at least one biomarker level selected under (a) in a second biological sample isolated from said pregnant female mammal, e.g. blood, plasma or urine, at a later time than said first sample; ;

(c) comparing the values measured in steps (a) and (b)

HbF, HbF and / or A1M level (s) in the second sample relative to the Hp-HbF, Hp, HO-1, Hpx, free HbF and / / Or the decrease in Hp, HO-1 and / or Hpx level (s)

Indicates that treatment is not effective as the PE progresses; A decrease in the Hp-HbF, HbF and / or A1M level (s) described above and / or an increase in Hp, HO-1 and / or Hpx level (s) indicate that the treatment is effective as the PE is degenerated.

More specifically, the method includes the following steps:

(a) I) Hpx, ii) Hpx and A1M, and iii) Hpx, A1M and HbF, respectively, in a first biological sample isolated from blood, plasma / serum or urine of pregnant female mammals before or during treatment. And optionally one or more biomarkers selected from one or more of Hp-HbF, Hp, HO-1,

(b) determining the level of one or more biomarkers selected under (a) from a second biological sample isolated from the pregnant female mammal, such as blood, plasma / serum or urine, at a later time than the first sample Measuring;

(c) Comparing the measured values in steps (a) and (b)

i) an increase in free HbF and / or A1M level (s) and, if appropriate, Hp-HbF, and / or Hpx levels and, if appropriate, levels of Hp, HO-1 (S) of

Indicates that treatment is not effective as the PE progresses;

Reduction of free HbF and / or A1M level (s), and, if appropriate, Hp-HbF levels; And / or Hpx levels, where appropriate, an increase in the Hp, HO-1 and / or Hpx level (s) described above indicates that the treatment is effective as the PE regresses.

In any of the above methods i) to ix), marker heme may also be included.

As mentioned above, preferred marker panels for use in predicting, diagnosing or assessing the risk of progression of PE according to the present invention are optionally supplemented with one or more of the following free HbF, Hp-HbF, Hp, HO- ≪ / RTI > Hpx and A1M.

In a specific embodiment, it is envisaged that the therapeutic efficacy can be assessed by determining whether the increase or decrease corresponds to a standard deviation of at least 1.1 or a 5% variance from a normal value as described above.

The invention also relates to a kit comprising a suitable reagent for the determination of an individual marker in a biological sample. Thus, the kit may contain an antibody to the individual marker, a means for performing an ELISA, or any other method referred to herein.

Materials and compositions for use in the prevention and / or treatment of preeclampsia

I) Ability to inhibit the formation of free Hb (free HbF or any other Hb), ii) Ability to inhibit the formation of free Hb (free HbF or any other Hb ), Or iii) any substance having the ability to reduce the concentration of free, cyclic free Hb (free HbF or any Hb) is potentially an effective potential for treatment and / or prevention of PE have. Thus, Hb binders; Heme binding / dissociation: iron-binding agent; Agents that stimulate hemoglobin degradation, heme degradation and / or iron blockade; And / or an agent that inhibits placental hematopoiesis for the treatment of PE.

Accordingly, in one aspect of the present invention, there is provided a method of treating a patient suffering from PE, comprising the steps of: predicting PE, diagnosing PE, evaluating the risk suffered by PE, The above-mentioned aspects can be supplemented by a therapeutic regimen involving the administration of one or more of the following substances to pregnant women.

More specifically, it is envisaged that the material is selected from i) to ix)

i) Antibodies or fragments thereof of hemoglobin

ii) Hoggle Robin

iii) CD 163

iv) Alpha-1-microglobulin

v) Hemopexin

vi) Heme-oxigenase

vii) albumin

viii) Transferrin

ix) Ferritin.

Hemoglobin-binding agent:

Antibody

Monoclonal antibodies with strong binding of Hb and blocking of the redox enzyme activity of Hb can be developed. The antibody may be produced by in vivo or in vitro immunization or is selected from an already existing library. Antibodies can be selected for specificity to the alpha-, beta-delta-or gamma-globin chain or to the common part of these globin chains. Antibodies can be modified to suit human therapy, i. E., Have a human immunoglobulin framework. Any portion of the antibody can be used: Fv-, Fab-fragment or whole immunoglobulin.

Hoggle Robin

Hp is a glycoprotein found in blood plasma / serum. There are three types of Hp (Hp1-1, Hp2-1 and Hp2-2). All forms bind to Hb and form Hp-Hb complexes. Hb-Hp complexes have a less potent redox enzyme activity than free Hb and thus cause less oxidative damage. Binding to Hb prevents, for example, iron loss from the heme group.

CD163

CD163 is a scavenger receptor found on macrophages, mononuclear cells, and the reticuloendothelial system, which form the blood vessel inner wall. Receptors recognize Hp-Hb complexes, mediate intracellular action and their transfer to lysosomes, breakdown by HO-1 (see below), and blockade of free iron by cellular ferritin. Thus, CD163 contributes to the elimination of Hb-induced oxidative stress.

Hem-Binder / Disintegration:

Hemopexin

Hpx is a glycoprotein (60 kDa) found in human blood plasma / serum, which is strongly bound to hem (Kd approximately 1 p mol / l) .

Hemoxygenase

Hemoxigenase is a heme-binding and degrading enzyme complex of cells that convert heme to biliruberidin, carbon monoxide and free iron. The latter is blocked by cellular ferritin, and bilirubin is reduced to bilirubin by bilirubin reductase, which is ultimately excreted in the urine. Three forms of the hemoxigenase gene (HO-1, HO-2 and HO-3) with very different structures have been described. HO-1 is the most important. These genes are upregulated in virtually all cells in the body by Hb, free heme, hypoxia, free radicals, ROS and a number of different inflammatory signals. HO-1 is a strong anti-oxidant because it not only removes oxidant heme and iron, but it also produces bilirubin, which has antioxidative effects on some oxidants.

albumin

Albumin is a 66 kDa protein in human blood plasma that can bind to hem. There is no evidence of cell uptake and degradation of the albumin-heme complex, and the effect of albumin acts as a repository of heme, thus potentially preventing the entry of heme into the endothelial cell membrane, vascular basement membrane, and the like.

Alpha-1-microglobulin

A1M is synthesized at a high rate in the liver, secreted into the bloodstream, and transported across the vessel wall to the extravascular segments of all organs. Proteins are also synthesized in other tissues (blood cells, brain, kidneys, skin), but at a lower rate. Due to its small size, the glass A1M is rapidly filtered from the blood in the kidneys. AIM is a general and specific superior anti-oxidation characteristic for oxidation, toxic degradation products of free Hb; Have properties that make them suitable for use in the treatment or prevention of various diseases accompanied by oxidative stress, or the presence of free Hb induces or aggravates the disease or condition.

A1M is an endogenous antioxidant that provides anti-oxidation in some ways. Thus, the present invention relates to A1M found to combine enzymatic reductase (Category 1), nonenzymatic reduction (Category 2) and radical-scavenging (Category 3) characteristics. In addition, the non-enzymatic reduction mechanism (Category 2) can be used repeatedly by several cycles of electron-donation. Furthermore, the radical-scavenger mechanism (Category 3) results in net production of electrons which further increases the anti-oxidizing capacity of the protein. In other words, a protein carries its own electron supply, is independent of the cell mechanism, and can act both intracellularly and extracellularly. In addition, A1M can restore oxidative damage associated with tissue components (a unique characteristic attributed to Category 4). See also the following for a detailed description of the radical scavenging mechanism.

A1M is a member of the lipocalin superfamily, a protein group from animals, plants, and bacteria that has a conserved three-dimensional structure but is highly functional. Each lipocalin consists of 160 to 190 amino acid chains that fold into a [beta] -barrel pocket with a hydrophobic interior. Twelve human lipocalin genes are known. Among human lipocalins, A1M is a 26 kDa plasma and is a tissue protein that is somewhat identified in mammals, algae and frogs. A1M is rapidly synthesized in the liver, secreted into the bloodstream, and transported rapidly across the vessel wall (T½ = 2 to 3 minutes) to the extravascular compartment of all organs. Proteins are also synthesized in other tissues (blood cells, brain, kidney, skin), but at a slower rate. A1M is found in the blood and in intestinal tissues as larger molecules (IgA, albumin, prothrombin) and in free, monomeric and covalent complexes. Due to its small size, the glass A1M is rapidly filtered from the blood in the kidneys. Subsequently, the major part is resorbed, but a significant amount is excreted in the urine.

Sequence and structural properties of A1M

The entire sequence of human A1M was first reported by Kaumeyer et al. (5). The protein was found to consist of 183 amino acid residues. Thereafter, at least 50 additional A1M cDNAs and / or proteins were detected, isolated and / or sequenced from other mammals, algae, amphibians and fish. The length of the peptide chain of A1M is slightly different between species, mainly due to mutations at the C-terminus. Sorting comparisons of different deduced amino acid sequences indicate that the percent identity varies from approximately 75-80% between rodents or placental mammals and humans, and approximately 45% between fish and mammals. At position 34 the free cysteine side chain is conserved. This group is involved in complex formation with other plasma proteins and in redox reactions in binding to the amber chromophore (see below). A computed 3D model based on the known X-ray crystallographic structure of other lipocalins suggests that Cys34 is a solvent exposed and positioned near the opening of the lipocalin pocket. Another lipocalin complementarity factor C8g also carries unpaired Cys at position 34 involved in the formation of the active C8 complex.

In the present context, the term "alpha-1-microglobulin" refers not only to SEQ ID NO: 1 (human A1M) but also to SEQ ID NO: 2 (human recombinant A1M), as well as to homologs, fragments or variants thereof having similar therapeutic activity Lt; RTI ID = 0.0 > alpha-1-microglobulin. ≪ / RTI > Thus, A1M as used herein is intended to mean a protein having at least 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. It is preferred that A1M as used herein has at least 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: It is even more preferred that A1M as used herein has at least 95%, such as 99% or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: In a preferred aspect, the alpha-1-microglobulin is according to SEQ ID NO: 1 or 2 as identified herein. In Fig. 10, a sequence listing of the amino acid sequences of human A1M and human recombinant A1M (SEQ ID NOs: 1 and 2, respectively) and the corresponding nucleotide sequence (SEQ ID NOs: 3 and 4, respectively) is given. However, the homologues, variants and fragments of A1M having important portions of the protein as identified below are also included in the term A1M as used herein.

As noted above, homologs of A1M may also be used in accordance with the description herein. In theory, A1M from all species, including the most primitive ones ever discovered, derived from fish (flounder) can be used. A1M is also available in isolated form from human, rat, mouse, rabbit, guinea pig, cattle and flounder.

It is important to note that even though A1M and bikunin have the same precursor, they have different amino acid compositions and have different properties. A1M belongs to the so-called lipocalin family, while Bikunin (also referred to as ulinastatin) belongs to the protease inhibitor superfamily.

Considering the homologues, variants and fragments of A1M, the following were identified as an important part of the protein for anti-oxidative effects:

Y22 (tyrosine, position 22, base pair 64 to 66)

C34 (cysteine, position 34, base pair 100 to 102)

K69 (lysine, position 69, base pair 205 to 207)

K92 (lysine, position 92, base pair 274 to 276)

K118 (lysine, position 118, base pairs 352 to 354)

K130 (lysine, position 130, base pair 388 to 390)

Y132 (tyrosine, position 132, base pair 394 to 396)

L180 (leucine, position 180, base pair 538 to 540)

I181 (isoleucine, position 181, base pair 541 to 543)

P182 (proline, position 182, base pair 544 to 546)

R183 (arginine, position 183, base pair 547 to 549)

(The numbering of amino acids and nucleotides as a whole refers to SEQ ID NOS: 1 and 3, and if other A1M, A1M analogs or recombinant sequences thereof from other species are used, those skilled in the art will be able to identify the active site (s) (S) of the amino acid sequence)

Thus, when A1M has, for example, 80% (or 90% or 95%) sequence identity to one of SEQ ID NO: 1 or 2, it is preferred that said amino acid is present at a suitable position in the molecule.

Human A1M is substituted with oligosaccharides in three positions, i.e., two sialylated complex types likely to be linked to two antenna carbohydrates to Asn17 and Asn96, and one or more simple oligosaccharides linked to Thr5. The carbohydrate content of the A1M protein from different species varies greatly, despite the absence of any glycosylation range in Xenopus leavis over a range of different glycosylation patterns.

A1M is yellowish brown when purified from plasma or urine. Colors are caused by heterogeneous compounds that are covalently linked to various amino acid bases located predominantly in the pocket entrance. These modifications are likely to represent oxidative degradation products of organic oxidants covalently linked by A1M in vivo, such as heme, kinurenine and tyrosyl radicals (6-8, 10).

A1M is also charge- and size-divergent, and the more highly brown A1M-molecules are more negatively charged. Possible explanations for heterogeneity are that they are changed to different degrees by different radicals and that the modifications alter the net charge of the protein. The covalently bonded color material was localized to Cys34, and Lys92, Lys118 and Lys130, the latter having a molecular weight of 100 to 300 Da. The tryptophan metabolite kininenin was found to be covalently attached to the lysyl residue in the A1M from the urine of hemodialysis patients, which in this case appears to be the source of the protein's yellow color (6). The oxidized fragment of the synthetic radical ABTS (2,2'-azino-di- (3-ethylbenzothiazoline) -6-sulfonic acid) is bound to the side chains of Y22 and Y132 (10).

C34 is the reaction center of A1M (9). This is highly negative, which has a high potential to provide electrons that are separated by the proximity of charged side chains to the amounts of K69, K92, K118 and K130, which is a prerequisite for sulfur atom oxidation, protein denaturation of the C34 thiol group . The preliminary data indicates that C34 is one of the most electronegative known groups.

Theoretically, the amino acids characterizing the unique enzymatic and non-enzymatic redox properties of A1M (C34, Y22, K92, K118, K130, Y132, L180, I181, P182, R183) Can be arranged in a framework, e. G., A protein with the same overall folding or a similar three-dimensional stereoscopic medium on fully artificial organic or inorganic molecules such as plastic polymers, nanoparticles or metal polymers.

Therefore, homologues, fragments or variants comprising a structure including the reaction center and its periphery as shown above are preferable.

Modifications and variations can be made in the structure of the polypeptides of this disclosure and still result in molecules (e.g., conservative amino acid substitutions) that have polypeptides-like characteristics. For example, a particular amino acid may substitute for another amino acid in the sequence without appreciable loss of activity. Because this is the mutual abilities and characteristics of the polypeptides that determine the biological functional activity of the polypeptide, certain amino acid sequence substitutions are made in the polypeptide sequence and nonetheless polypeptides with similar properties can be obtained.

In making this change, the hydropathic index of the amino acid can be considered. The importance of the hydrophobic amino acid index in conferring a mutual biological function on a polypeptide is generally understood in the art. It is known that certain amino acids substitute for other amino acids with similar hydrophobicity indexes or scores, and may still result in polypeptides having similar biological activities. Each amino acid was given a hydrophobicity index based on its hydrophobicity and charge characteristics. The indices are: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alinine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The relative hydrophobic character of the amino acid determines the secondary structure of the resulting polypeptide, which ultimately determines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is well known in the art that amino acids can be substituted by other amino acids having similar hydrophobicity indices and still functionally equivalent polypeptides can be obtained. In such a change, the substitution of the amino acid whose hydrophobicity index is within ± 2 is preferred, the substitution within ± 1 is particularly preferred, and the substitution within ± 0.5 is even more particularly preferred.

Substitutions of similar amino acids based on hydrophilicity can also be made, particularly such biologically functional polypeptides or peptides thus generated are intended for use in immunological embodiments. The following hydrophilicity values were assigned to the amino acid residues: arginine (+3.0); Lysine (+3.0); Aspartate (+3.0 + -1); Glutamate (+3.0 + -1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Proline (-0.5 ± 1); Threonine (-0.4); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). It is understood that amino acids can replace others with similar hydrophilicity values, and still be biologically equivalent, particularly immunologically equivalent polypeptides. In such a change, substitution of an amino acid whose hydrophilicity value is within ± 2 is preferred, substitution within ± 1 is particularly preferred, and substitution within ± 0.5 is even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that consider one or more of the characteristics mentioned above are well known to those skilled in the art, (original residue: exemplary substitution): (Ala: GIy, Ser), (Arg: Lys), (Asn: GIn ₁ (GIu: Asp), (GIy: Ala), (His: Asn, Gin), (Ile: Leu, VaI), (Leu: Asp) (Tyr: Trp, Phe), and (VaI: Lle (Ile, VaI), (Lys: Arg), (Met: Leu, Tyr) , Leu). ≪ / RTI > Embodiments of the present disclosure thus contemplate functional or biological equivalents of the polypeptides as set forth above. In particular, embodiments of the polypeptides may include variants having about 50%, 60%, 70%, 80%, 90% and 95% sequence identity to the polypeptide of interest.

In the present context, homology between two amino acid sequences or between two nucleic acid sequences is described by the parameter "identity ". Alignment of sequences and calculation of homology scores can be done using a full Smith-Waterman alignment that is useful for both protein and DNA alignment. The default scoring matrix BLOSUM50 and identity matrix are used for each of the protein and DNA alignments. The penalty for the first residue in the gap is -12 for protein and -16 for DNA while the penalty for additional residues in the gap is -2 for protein and -4 for DNA. Sorting can be done by FASTA package version v20u6.

Multiple alignments of protein sequences can be achieved using "ClustalW ". Multiple alignments of the DNA sequence can be made using protein alignment as a template to replace the amino acid with the corresponding codon from the DNA sequence.

Alternatively, different software can be used to align the amino acid sequence and the DNA sequence. Alignment of the two amino acid sequences is determined, for example, by using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the overall sorting algorithm described. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5.

The degree of identity between amino acid sequences; For example, SEQ ID NO. 1 and a different amino acid sequence (e. G., SEQ ID NO. 2) can be used to determine the exact number of matches in the alignment of two sequences to either the length of SEQ ID NO. 1 or the length of SEQ ID NO. Calculate by dividing by short. The results are expressed as percent identity.

Accurate matching occurs when two sequences share the same amino acid organs at the same position.

If appropriate, the degree of identity between the two nucleotide sequences can be determined using the LASER-GENE MEGALIGN software (DNASTAR, Inc., Wisconsin, Wis.) By the identity table and the following multiple alignment parameters (gap penalty 10 and gap length penalty 10) Wilmington, Del., Madison, Wis.). The pairwise alignment parameters are Ktuple = 3, gap penalty = 3, and window = 20.

In certain embodiments, the percentage identity of the amino acid sequence of the polypeptide to or against the amino acid sequence of SEQ ID NO: 1 is determined by: i) comparing the two amino acid sequences using the Needle program with the LOSUM62 substitution matrix, gap opening penalty 10, and gap extension penalty 0.5 Sort; ii) digitize the exact number of matches in the alignment; iii) dividing the exact number of matches by the length of the shorter of the two amino acid sequences, and iv) converting the result of dividing iii) to a percentage. Percent identity to other sequences of the invention or to other sequences is calculated in a similar manner.

By way of example, a polypeptide sequence may be the same for 100% identical baseline sequences, or may include up to a certain integer number of amino acid changes relative to a baseline sequence such that percent identity is less than 100%. Such modifications are selected from at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, wherein the alteration is made at the amino- or carboxy-terminal position of the reference polypeptide sequence or in the amino acid Can occur either individually or between the corresponding terminal positions located in one or more adjacent groups within the reference sequence.

Conservative amino acid variants may also include non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4- hydroxyproline, N- Hydroxy-ethyl cysteine, hydroxyethyl homocysteine, nitro-glutamine, homoglutamine, piperol acid, thiazolidinecarboxylic acid, dehydroproroline, 3- and 4- methylproline, 3,3- , Norvaline, 2-azaphenyl-alanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, in vitro systems may be used, but nonsense mutations are inhibited using chemically aminoacylated inhibitory tRNAs. Methods for synthesizing amino acids and aminoacylation tRNAs are well known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. In a second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated inhibitory tRNA. In a third method, the E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and the desired non-naturally occurring amino acid (s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, - < / RTI > azaphenylalanine or 4-fluorophenylalanine). The non-native amino acid is incorporated into the protein instead of its natural counterpart. Naturally occurring amino acid residues can be converted to non-naturally occurring species by chemical transformation in vitro. Chemical modifications can be combined with site directed mutagenesis to further expand the range of substitution. An alternative chemical structure that provides a sufficient three-dimensional structure to support the antioxidant properties of A1M may be provided by other techniques, such as artificial scaffolding, amino acid substitution, and the like. Furthermore, it is assumed that the structure mimicking the above-mentioned active sites of A1M has the same function as A1M.

Iron-Binder:

Transferrin

Transferrin is the most important transporter of iron in the blood. Transferrin-iron complexes are recognized and bound by receptors in the cell that internalize and dissociate the complex.

Ferritin

This multimeric protein, consisting of 24 subunits of both types, is a major intracellular depot of free iron. It has a high iron-storage capacity, i.e., 4500 iron atoms / ferritin molecules. Based on ferritin, iron is largely prevented from oxidizing and reducing reactions, thus preventing oxidation damage.

In a further embodiment, the Hb binding agent is an antibody specific for Hb and / or heme.

In a specific embodiment, the pharmaceutical formulation comprises a combination of Hb binder and / or heme binder and / or iron sequestrant.

Agents that stimulate Hb degradation and / or heme degradation include, but are not limited to, proteins such as Hp, Hpx, and HO.

Pharmaceutical preparations containing one or more of the above-mentioned substances may be administered to a "placenta" animal such as a human, other primate, or mammalian food animal. Preferred animals for administration are human or commercially valuable animals or livestock.

Administration may be carried out in different ways depending on the animal being treated, the condition of the animal in need of treatment, and the specific indication for treatment. The route of administration may be oral, rectal, parenteral, or through the nasogastric tube under conditions such that the active agent can be delivered to the fetal environment, such as fetal placental circulation, amniotic fluid, and the like. Parenteral administration is preferred. Examples of parenteral administration routes are intravenous, intraperitoneal, intramuscular or subcutaneous injection.

The formulation of the pharmaceutical preparation should be selected according to the pharmacological properties of the active ingredient as well as its physicochemical properties and type of route of administration. Different methods of formulating pharmaceutical preparations are well known to those skilled in the art.

In general, pharmaceutical compositions comprising A1M (or analogs, fragments or variants thereof as set forth herein) or any other substance referred to herein may be used in i.v. Lt; / RTI > Thus, the material may be formulated in a liquid, for example, as a solution, dispersion, emulsion, suspension or the like. As is evident from the examples herein, i.v. A suitable vehicle for administration may consist of 10 mM Tris-HCl, pH 8.0 and 9.125 M NaCl.

Suitable solvents for parenteral use include water, alcohols, lipids, vegetable oils, propylene glycol, and organic solvents generally approved for this purpose. Generally, one of ordinary skill in the art will refer to Gennaro et al. For example, in Remington's Pharmaceutical Science edited by Mack Publishing Company and in Handbook of Pharmaceutical Excipients edited by Rowe et al. (PhP Press), and on the appropriate excipient for the specific formulation type, (E.g., Ph.Eur. Or USP) on methods for making a < / RTI > Suitable excipients include, but are not limited to, solvents (e.g., water, aqueous media, alcohols, vegetable oils, lipids, organic solvents such as propylene glycol), osmotic pressure regulators (e.g., sodium chloride, mannitol, etc.), solubilizers, (If appropriate), absorption enhancers, and the like.

The substance may be administered in one or several doses with respect to the dose of the radionuclide therapy. Preferably, each dose will be administered as a single dose, i.v. as a slow dose only after a single dose, for a short duration up to 60 minutes, or as a slow dose for a short duration up to 60 minutes. Additional capacity may be added. When A1M is used, each dose contains the amount of AlM associated with the patient's body weight: 1 to 15 mg A1M / kg patient.

For oral compositions, the compositions may be in solid, semi-solid, or liquid form. Suitable compositions include solid formulations (e.g., tablets, including all kinds of tablets, sachets and capsules), powders, granules, pellets, beads, syrups, mixtures, suspensions, emulsions and the like.

Suitable excipients include, for example, fillers, binders, disintegrants, lubricants, etc. (for compositions in solid form or solid form), liquids or semi-solid forms for solvents such as water, organic solvents, vegetable oils and the like . In addition, additives such as pH adjusting agents, taste masking agents, flavoring agents, stabilizers and the like may be added.

In addition, certain carriers may be included for targeting the active material to a specific portion of the body. Antibody-A1M complex in which the antibody is targeted to the placenta by its specificity to the placenta-epitope ("oesophage"); Recombinant cells having stem cell or placenta-specific characteristics, for example, an integrin-receptor having an artificial or natural ability to secrete a large amount of A1M specific to the placenta. Treatment will be more effective because the drug is concentrated in the placenta.

The term "effective amount" in the context of the present invention refers to an amount that provides a therapeutic effect for a given condition and dosage regimen. This includes the necessary additives and diluents; That is, a predetermined amount of active substance calculated to produce the desired therapeutic effect in association with the carrier or administration vehicle. In addition, it is intended to mean an amount that is abundant in reducing and most preferably preventing clinically significant deficiencies in the activity and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in the clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of compound will vary depending on its particular activity. Suitable dosages include the intended diluent; That is, it may contain a predetermined amount of the active composition calculated to treat the desired therapeutic effect in association with the carrier or additive. In addition, the dosage to be administered will generally be in the range of 0.001 to 1000 mg / kg body weight per day, depending on the active principle or principle to be used, the age, body weight, etc. of the patient to be treated. In addition, it will depend on the route of administration of the dose.

Figure 1. Correlation between cell-free HbF- and Hp concentrations - Samples were from normal pregnant (control) and women diagnosed with PE. The cell-free HbF plasma concentration of each patient sample (control and PE) was plotted against Hp plasma concentration ( A ). The plasma-free HbF plasma concentration of the control was plotted against Hp plasma concentration ( B ). Cell-free HbF plasma concentrations of women diagnosed with PE were plotted against Hp plasma concentrations ( C ). The association between values is linear regression analysis (Pearson). (Research I)
Figure 2. Hp isoforms, acellular HbF- and Hp-HbF levels - The correlation between Hp-isoforms (1-1, 1-2 or 2-2) was determined by SDS-PAGE and Western blot analysis by anti-Hp antibody as shown in the three patient examples as described in Materials and Methods ( A ) The distribution of the different isoforms is presented as the average percentage of women in Hp 1-1, 1-2 and 2-2 for each group ( B ). Plasma concentrations of cell-free HbF ( C ) and Hp-HbF ( D ) are shown separately in patient samples with Hp isoforms (Hp 1-1, 1-2 and 2-2), respectively. The results are presented as the average percentage of each Hp isoform (Hp 1-1, 1-2 and 2-2) in B. Results are presented as mean ± SEM plasma concentrations of cell-free HbF and Hp-HbF in C and D (Study I).
Figure 3. Correlation between Hpx concentration and systolic / diastolic blood pressure - The highest systolic ( A ) and diastolic ( B ) blood pressures (BP) measured within the last two weeks prior to transmission were plotted against plasma concentrations of Hpx. (Research I)
Figure 4. Receiver operating characteristic (ROC) curves - ROC curves showing sensitivity and specificity for combinations of HbF, A1M and Hpx ( A ), Hpx and A1M ( B ) and Hpx ( C ). The area under the curve (AUC) is 0.88 for a combination of HbF, A1M and Hpx, 0.92 for a combination of A1M and Hpx, and 0.87 for Hpx. (Research I)
Figure 5. Schematic representation of a potential chain of events involving HbF, Hp, Hpx, A1M, and ROS, with a schematic representation of the potential placenta with impaired fetal-maternal barrier function leading to leak of placental factor . 1: Placental early events induce upregulation of placental HbF gene and protein and ROS. 2: Oxidative damage and leakage of fetal-maternal barriers lead to increased maternal plasma concentrations of HbF. 3: Excess oxy Hb undergoes a self-oxidation reaction leading to formation of free heme-group and ROS. 4: The complex network of scavenger proteins consisting of Hp, Hpx and A1M binds, inhibits, and eliminates HbF, heme and ROS. Cell-free HbF is bound by Hp and is clear by CD163 receptor-mediated absorption in monocytes and macrophages. The free heme-group is bound by Hpx, and the heme is preferably clear through the Hpx receptor CD91 expressed on macrophages and hepatocytes. In this study, a highly significant reduction in both Hp and Hpx was observed in maternal plasma of women with PE compared to normal pregnancy. Indicating an increased level of prolonged presence of both extracellular Hb and heme. Analysis of plasma A1M levels in this study showed a significant increase in women with PE compared to normal pregnancies, most likely as a result of oxidative stress-induced upregulation of A1M gene expression.
Figure 6. Recipient Manifestation Characteristics (ROC) curves - recipient manipulation curves for combinations of HbF, A1M haemopectin, haptoglobin and these biomarkers as predictive biomarkers for all PEs. Specific values are found in Table 10. (Study II)
Figure 7. Receiver Manipulation Feature (ROC) curve - recipient manipulation curve of a combination of maternal features and biomarkers and maternal features as markers of all PEs. Specific values are found in Table 10. (Study II)
Figure 8. Correlation between Hpx30 and diastolic blood pressure - Correlation between Hpx30 and diastolic blood pressure in all patients. Specific values are found in Table 5. (Research I)
9. Logistic regression analysis - The combination of HbF, Hpx activity, Hpx concentration, heme and HO-1 showed 10% false positive rate (FPR) and 84% DR at AUC 0.93.
10. Sequence Listing
Figure 11. Level of A1M compared to the control group in different gestational age.

In this study, we used PE as a model disease to study the response of a cell-free Hb-defensive network in pathological conditions due to prolonged elevation of hemolysis. Therefore, in order to study the physiological relevance and possible pathophysiological significance of cell-free HbF in disease progression of PE, the present inventors investigated the effect of cell-free (Hp, Hpx, A1M and CD163) on the major human endogenous Hb-scavenging systems The effects of HbF (both in free (indicating HbF) and in Hp (Hp-HbF)) were studied. This allowed the present inventors to study the potential for HbF, Hp-HbF, Hb-whole, A1M, Hp, Hpx and CD163 as biochemical markers supporting the diagnosis of PE.

In this study, the present inventors characterize acellular HbF and endogenous Hb- and hem-scavenger systems at scheduled dates in pregnant women diagnosed with PE and normal pregnancy (control). Similar to the previous results, the present inventors have found a significant increase in HbF in women with PE ¹¹ . Furthermore, the plasma levels of the Hb- and heme scavenger systems are highly affected and represent a significantly reduced level of scavenger Hp and hem-scavenger Hpx. Interestingly, and according to previously published studies, the extravascular heme- and radical scavenger A1M was significantly increased in plasma of women with PE.

In this study, the present inventors also evaluated the diagnostic and clinical utility of the biomarkers studied and found a clear potential for using them as clinical tools in diagnosing women with PE and / or HELLP. Furthermore, biomarkers have shown clinical utility, which has made it possible to identify women and fetuses at risk for clinical complications.

Hemolysis and subsequent release of cell-free Hb and heme occurs in a wide variety of clinical conditions and diseases, including HELLP syndrome, transfusion reactions, malaria, hemorrhage, sepsis and sickle cell disease. The release of acellular Hb and heme results in a variety of pathophysiological effects that constitute a major seizure, hemodynamic instability and tissue damage. Immediate effects include scavenging of strong vasodilator nitrogen monoxide (NO) which causes increased arterial blood pressure. Further, acellular Hb and free heme accumulate in the vessel wall and organ and are compartmentalized, resulting in subsequent organ failure. Importantly, prolonged exposure to cell-free Hb and heme has been described as associated with NO depletion, inflammation and oxidative stress.

In a recent series of publications, the implications and significance of the pathogenesis of cell-free HbF and its downstream metabolite free hemoglobin and ROS in the progression of PE-associated impairment and syndrome have been characterized. Using a double placental perfusion system, May et al. Have shown that the addition of cell-free Hb to the fetal circulation is associated with a significant increase in perfusion pressure, fetal-maternal leaking of extracellular Hb into maternal circulation, Resulting in morphological impairment. Using the pregnant lamb PE-model and the pregnant rabbit model, the starvation state resulted in increased amounts of extracellular heme and bilirubin in haemolysis and blood. Furthermore, in these models, severe placenta and kidney damage have also been described. This damage was thought to be caused by increased hemolysis and subsequent release of Hb and by the production of heme and ROS.

In this disclosure, we have found that, in connection with previous studies, cell-free HbF (both HbF and Hp-HbF) was significantly increased in pregnant women diagnosed with PE. Thus, these women have increased blood pressure and protein leakage into the urine, both of which are characteristic of pathological physiological exposure to acellular Hb and heme.

To protect itself against extracellular Hb and free heme, humans have evolved some Hb- and heme-detoxification systems. The best studied Hb-scavenger system is Hp. Hp binds to extracellular Hb in the blood very efficiently, and the resulting Hp-Hb complex is cleared from the blood by binding to the macrophage receptor CD163. If Hp is abundantly removed or exhausted as a result of prolonged exposure to Hb, excess oxy Hb will undergo a self-oxidation reaction leading to free heme-group and ROS. Moreover, excess and non-protein binding will lead to the leakage of protein into the Hb is accumulated in the kidney causes damage to the kidney, subsequently the urine ^46. Depletion, depletion, or overwhelm of Hp will cause the oxy Hb to break down into its metabolites metHb, free heme and ROS. The main hem-scavenger in the bloodstream is Hpx, a highly specific and common system for protecting cells, blood vessels and tissues against heme-induced damage. After binding, Hpx transfers heme through its receptor CD91, which is preferably expressed on its macrophages, hepatocytes, neurons, and haploid trophoblasts, which are internalized by receptor-mediated inclusion and hems are subsequently degraded.

In this study, a highly significant reduction in both Hp and Hpx was observed in maternal plasma of women with PE compared to normal pregnancy. Indicating an increased level of prolonged presence of both extracellular Hb and heme. Thus, although there is no very high level of cell-free HbF, we have found that continuous exposure to low or moderate levels from early pregnancy (for example, studies by Dolberg et al. Suggesting cell HbF depletion) endogenous vascular Hb- and heme (i.e., Hp and Hpx) protection systems. In addition, in some PE patients, we observed a highly significant increase in cell-free HbF (non-Hp-binding). Very interestingly, all these patients found to be Hp 2-2 isoform (FIG. 2C). Thus, these subgroups of PE patients could have reduced innate defense against acellular Hb, and indeed could constitute a high-risk population.

The present inventors have already shown that the radical scavenger A1M binds to and degrades heme and protects cells and tissues from damage to oxidation, mitochondria, cell and tissue structure and cell death. Furthermore, the inventors have shown that plasma A1M levels are significantly increased in women with PE at both the end of the gestation period and in the early gestation. Analysis of plasma A1M levels in this study identified previously published data showing a significant increase in A1M plasma concentrations in women with PE compared to normal pregnancies.

Why is the A1M-level increased in PE patients while the Hp- and Hpx- levels are reduced? It has been reported that A1M gene expression is rapidly up-regulated in the liver, skin, placenta and other organs as a response to increased levels of Hb, heme and ROS. Which will result in an increased secretion of protein resulting in increased plasma concentrations in pathological conditions with increased Hb and ROS load. Furthermore, the A1M specific receptor-mediated clearance system has not been shown to be triggered during hemolysis or oxidative stress, while Hp and Hpx are clear from plasma upon binding to Hb and heme. As a result, the concentration of A1M in the plasma and extravascular fluid will be increased, whereas Hp and Hpx will be depleted, and therefore their plasma concentration will be reduced.

There is an increased focus on the use of biomarkers in clinical prediction and diagnosis of PE. Although some biomarkers have been presented so far, there were no available guidelines recommending the use of biomarkers in clinical situations. Recently, the American Congress of Obstetricians and Gynecologists (ACOG) has reported that proteinuria is associated with platelet (<100,000 / microliter), serum creatinine (> 1.1mg / dl) and liver transaminase The definition of severe PE replaced by the use of the included biomarkers is presented. Herein, we present data suggesting that the biomarkers HbF, A1M and Hpx can be used clinically to support the diagnosis of PE. The combination of Hpx and AlM showed the highest correlation (5% false positive, 69%, AUC = 0.88, Figure 4A) and the combination of Hpx and A1M also showed a high detection rate (5% 66% in false positive, AUC = 0.87, Fig. 4B). Thus, HbF, Hpx and A1M constitute possible future markers that can support the diagnosis of PE.

Hpx concentrations were found to have a significant negative correlation to blood pressure, i. E. Severity of disease (FIG. 3). Previous studies have shown that active Hpx can affect the renin-angiotensin system (RAS) in vitro by downregulating the angiotensin II receptor (AT (1)) and promoting expanded blood vessel morphology ^{53 , 54} . Increased acellular HbF levels in women with preeclampsia could lead to increased consumption of Hpx and consequently reduced Hpx activity leading to improved AT (1) receptor expression and contracted blood vessel morphology. In fact, Bakker et ^al.54 showed that plasma from women with preeclampsia had increased AT (1) receptor expression on mononuclear cells compared to plasma from normal pregnancies. This may be an important blood pressure control effect caused by elevated extracellular HbF observed in PE with NO consumption.

Being able to predict fetal and maternal outcomes has tremendous clinical value because it can help clinicians in difficult tasks optimizing delivery times. In this study, we evaluated the correlation between the biomarkers studied and various maternal and fetal outcomes. The results indicated that HbF, Hp and Hpx correlated with admission to NICU. Further, Hpx was strongly associated with premature birth. However, since all prematurity in this cohort is associated with preeclampsia, this strong association could be a result of a strong correlation between Hpx and preeclampsia rather than prematurity itself.

It is important to note that the cohort-control study used in this case is not a normal distribution cohort, i. E. It includes overrepresentation of women with PE. As a result, the detection and prediction rates reported in this study could therefore be different in a normal distribution cohort containing 3-8% of PE cases.

In this study, we have another cell characterized by acellular HbF and endogenous Hb- and hem-scavenger in pregnancy with complications due to preeclampsia. Plasma levels of HbF were significantly elevated, while Hp and Hpx were significantly reduced in women with preeclampsia. A1M, an extravascular hem- and radical scavenger, and a marker of oxidative stress, was significantly increased in the plasma of preeclampsia. Furthermore, HbF and related scavenger proteins have shown potential to be used as clinical biomarkers for more accurate diagnosis of preeclampsia and as predictors to assist in the identification of pregnancies with increased risk of acid and complications.

In this study, HO-1 concentrations were significantly reduced, especially in late-onset PE groups. Low concentrations of HO-1 could be attributed to continued deformation of the system due to elevated heme and HbF levels throughout the PE. HO-1 enzyme is gradually depleted throughout pregnancy, and thus lower in late-onset PE.

Plasma heme concentrations were both elevated in early and late onset PE, but only significantly elevated in late onset PE. The heme concentration was clearly correlated with the total Hb concentration. Previously published studies have shown that increased levels of HbF throughout the PE pregnancy slowly de-act and deplete the maternal Hb and hemspabeling system, including A1M, haptoglobin and hemopexin concentrations. Constant overgrowth of placental HbF induces damage to the placenta and maternal endothelium. The strength of the maternal scavenger and enzyme system may be an important component in determining how and when clinical symptoms are present in stage 2 of PE. The more systems there are, the deformation and / or depletion, and the more severe the symptoms are.

Correlation analysis showed a significant inverse correlation between Hpx activity and diastolic blood pressure in all patients.

Hemoxigenin 1 also had an inverse correlation with systolic and diastolic blood pressures. The higher the heme load, the lower the HO 1 in PE patients. The depletion of HO-1 weakens the anti-inflammatory properties, which can eventually exacerbate the maternal endothelium, thus increasing blood pressure. Furthermore, decomposition of heme by HO-1 produces a strong vasodilator, CO. The reduced level of HO-1 results in reduced degradation of heme and less production of CO. This could be added to the contacted blood vessels seen in patients with PE.

In this study, we present various potential biomarkers based on HbF and hemoglobin- and hemoscavenger proteins and enzymes. The biomarkers used in combination reach an acceptable level of detection that is acceptable for clinical use. As a single marker, Hpx activity was able to detect 30% of PE cases at 10% FPR. Heme and HO-1 showed similar DR. Taken together, however, Hpx activity, Hpx, HO-1, heme and HbF concentrations can detect 84% of PE cases at 10% FPR, which matches some of the best biomarkers in PE. Furthermore, some of the biomarkers included in the model presented are correlated with blood pressure, and therefore correlated with clinical severity of PE.

By measuring the Hb metabolite component as a potential diagnostic biomarker, a more accurate PE diagnosis can be achieved.

Experiment

Materials and methods

Study I - Sampling at 34 to 40 weeks of pregnancy

Patient and Demographic

At the start, 150 pregnant women were included in the study. Patients were randomly and retrospectively selected from current ongoing cohort studies. Exclusion criteria are hypertension during pregnancy, essential hypertension, and gestational diabetes. A total of 145 cases were excluded due to diabetes or pregnancy-related diabetes mellitus in 5 cases in total, and a total of 145 patients included 98 progressive PE (cases) and 47 normal pregnancies (control). Patient demographics are listed in Tables 1 and 2.

Sample collection

This study was approved by the Ethics Committee for the Study of Human Subjects at Lund University in Sweden. After verbally and in writing, the patient signed a preliminary agreement. Maternal venous samples were taken from the patients who were certified for obstetrics and gynecology at Lund University Hospital in Sweden before delivery. Samples were collected as 6 ml of blood in an EDTA Vacuette (TM) plasma tube (Greiner Bio-One GmbH, Kremsmünster, Austria) and then centrifuged at 2000 xg for 20 minutes. Plasma was then transferred into the freezing tube and stored at-80 C until assay time. The pregnancy outcomes for each patient were taken from the chart backwards.

Preeclampsia was defined as de novo hypertension by two separate readings of blood pressure ≥140 / 90 mmHg and proteinuria ≥300 mg for at least 4 hours per 24 hours after 20 weeks of gestation. This is in accordance with the International Society for Hypertension Research in the definition of pregnancy ⁵⁰ . Without quantification of proteinuria, dipstick analysis was allowed. Further, the PE group was further subcategorized as early onset PE (diagnosis ≤ 34 + 0 weeks of gestation) or late onset PE (diagnosis> 34 + 0 weeks of gestation). There were three cases of PE due to unknown diagnosis time and therefore did not include the analysis made by the initial PE and late PE PE subgroups.

Reagents and proteins

에 의해 기재한 바와 같이 소변으로부터 정제하였다⁵⁷. 토끼 다클론성 항체를 인간 A1M에 대해 제조하였고⁵⁸, 인간 A1M에 대한 마우스 단클론성 항체⁵⁹, 염소 항-인간 A1M 및 염소 항-토끼 면역글로불린을 앞서 기재한 바와 같이 제조하였다⁶⁰.HbF was purified as described previously from the whole blood drawn from the umbilical cord blood ¹⁶ newly. Human γ- chain p- Murray kyure benzoate ⁵⁶ with due to dissociation of the purified HbF and Noble strain using (Sigma-Aldrich St. Louis, Missouri, in the United States) Document [Kajita et al.] Was prepared by acid precipitation as described by ^55. Absolute purity of HbF (from contamination by HbA) and γ-chain (from contamination by α- and β-chains) was determined as described previously ¹¹ . Mouse antibodies to human γ-chain, and thus specificity for HbF, were generated and purified by AgriSera AB (Banannas, Sweden). Anti-HbF antibodies were conjugated with mustard radish peroxidase (Lightning-Link HRP, Innova Biosciences, Cambridge, UK) as described by the manufacturer. The human A1M

Lt; RTI ID = 0.0 &gt; ⁵⁷ &lt; / ^RTI &gt; Rabbit polyclonal antibodies were prepared for human A1M ⁵⁸ , mouse monoclonal antibody ⁵⁹ for human A1M, goat anti-human A1M and goat anti-rabbit immunoglobulin were prepared as previously described ⁶⁰ .

Fetal hemoglobin ( HbF )-density

A sandwich-ELISA was used to quantify non-complex HbF. The 96-well microtiter plates were coated with anti-HbF antibody (mouse monoclonal, 4 [mu] g / ml in PBS) at room temperature (RT) overnight. In the second step, the wells were blocked with blocking buffer (1% BSA in PBS) for 2 hours and then incubated with the HbF calibrator or patient sample at room temperature for 2 hours. In the third step, HRP-conjugated anti-HbF antibody (mouse monoclonal; diluted 1: 5000) was added and then incubated for 2 hours at RT. Finally, ready-to-use 3,3 ', 5,5 "-tetramethylbenzidine (TMB, Life Technologies, Stockholm, Sweden) substrate solution was added. After 20 minutes with 1.0 M HCl, the reaction was stopped and the absorbance was read at 450 nm using a Wallac 1420 multi-label counter (Perkin Elmer Life Sciences, Waltham, Mass., USA).

Hoggle Robin - fetal hemoglobin (Hp- HbF ) Concentration

A sandwich-ELISA was used for the quantification of Hp-HbF. This ELISA shows a high preference for Hp-HbF compared to uncomplexed HbF (more than 10x of the Hp-HbF calibrator series compared to the HbF calibrator series at the same molar content of HbF). A 96-well microtiter plate was coated with anti-Hp-HbF antibody (HbF-affinity purified rabbit polyclonal: 4 ug / ml in PBS) overnight at room temperature. In the second step, wells were blocked with blocking buffer (1% BSA in PBS) for 2 hours and then incubated with Hp-HbF calibrator or patient sample at room temperature for 2 hours. In the third step, an HRP-conjugated anti-Hb antibody (HbA-affinity purified rabbit polyclonal, diluted 1: 5000) was added and then incubated at room temperature for 2 hours. Finally, the ready-to-use TMB (Life Technologies) substrate solution was added and the reaction was stopped after 30 minutes with 1.0 M HCl and absorbance at 450 nm using a Wallac 1420 multi-label counter (Perkin Elmer Life Sciences) .

Total hemoglobin (Hb-total) -density

 Human Hb ELISA quantitation kit from Genway Biotech Inc. (San Diego, CA, USA) was used to determine total Hb-concentration in maternal plasma. After performing the analysis according to the manufacturer's instructions, the absorbance was read at 450 nm using a Wallac 1420 multi-label counter.

Alpha-1-microglobulin ( A1M )-density

Radioimmunoassay of A1M was performed using ¹²⁵ I (Perkin Elmer Life Sciences) using the chloramine T method. Protein-bound iodine was separated from free iodine by gel-chromatography on a Sephadex G-25 column (PD10, GE Healthcare, Stockholm, Sweden). A specific activity of approximately 0.1 to 0.2 MBq / 占 퐂 protein was obtained. Radioimmunoassay (RIA) was performed by mixing chlorine antisera for human A1M (1: 6000 dilution) with ¹²⁵ I-labeled A1M (approximately 0.05 pg / ml) and an unknown patient sample or calibrator A1M- concentration. After overnight incubation at RT, antibody-binding antigen was precipitated by addition of bovine serum and 15% polyethylene glycol, followed by centrifugation at 2500 rpm for 40 minutes, after which the ¹²⁵ I-activity of the pellet was measured by Wallac Wizard 1470 gamma Counter (Perkin Elmer Life Sciences).

Hoggle Robin  (Hp) concentration

Human Hp ELISA quantitation kit from Genway Biotech Inc. was used to determine Hp concentrations in maternal plasma. After performing the analysis according to the manufacturer's instructions, the absorbance was read at 450 nm using a Wallac 1420 multi-label counter.

Hemopexin  ( Hpx )-density

Human Hpx ELISA kits from Genway Biotech Inc were used to determine Hpx concentrations in maternal plasma. The assay was performed according to the manufacturer's instructions, and then the absorbance was read at 450 nm using a Wallac 1420 multi-label counter.

Hpx activity

Plasma Hpx activity was measured in an EDTA plasma sample using an Hpx-MCA substrate (synthesized by Pepscan of Holland Lelystat). Plasma samples (40 μl) were diluted 1: 4 in a final volume of 200 μl using a substrate solution (0.2 M Tris + 0.9% NaCl pH 7.6 (substrate concentration 80 μM / L) at 37 ° C. Varioskan spectrophotometer The Hpx activity was measured at 0 min, 30 min (Hpx30), 60 min (Hpx60) and 24 hrs after the measurement of the total amount of serine migrated by Hpx at a given time If the value after the 24 hour incubation was less than 5, the activity was considered to be very low due to technical problems due to analysis or samples and the sample was excluded from further analysis. The curve shows that the inverse analysis is Hpx30 and Hpx60 measurements (HpxAUC Measurements Hpx30, Hpx60, and HpxAUC were similar, so only Hpx30 was used for analysis. We refer to Hpx activity in the following Hpx30.

Cluster of differentiation 163 (CD163) - Concentration

The human CD163 duo set from R & D R & D Systems (Abingdon, UK) was used to determine the concentration of CD163 in maternal plasma. After performing the analysis according to the manufacturer's instructions, the absorbance was read at 450 nm using a Wallac 1420 multi-label counter.

SDS-PAGE and Western Blot

SDS-PAGE was performed using precast 4-20% mini-protein TGX gels from Bio-Rad (Hacillis, CA) and then the molecular weight standards (precision protein + double marker Bio-Rad) , Under reducing conditions. The separated proteins were transferred to polyvinylidene difluoride (PVDF) or low fluorescence (LF) PVDF membranes (Bio-Rad). The membranes were then incubated with antibodies against Hp (polyclonal rabbit-anti-human Hp, 12? G / ml, DAKO in Glostrup, Denmark). Western blotting was performed using HRP-conjugated secondary antibody (DAKO) and chemiluminescent substrate Clarity Western ECL (Bio-Rad). The band was detected in the ChemiDoc XRS unit (Bio-Rad). Relative quantification of the A1M band was performed by densitometry using Image Lab software (Bio-Rad).

Statistical analysis

To analyze the data, statistical computer software statistical package for social science (SPSS Inc., Chicago, IL, USA) version 21 for Apple computers (Apple Inc., Cupertino, Calif.) And Origin 9.0 software OriginLab Corporation, Northampton) was used.

The ANOVA test was used to compare groups for clinical parameters such as age, BMI, equivalence, systolic blood pressure, diastolic blood pressure, proteinuria, pregnancy at delivery, birth weight, sampling at 10 min and sampling time at APGAR .

Hpx activity, Hpx, HO-1, heme, HbF and PE were compared using the Mann-Whitney test. Subgroup-analysis was performed for early- and late-onset PE.

Chi square tests were used to compare groups for fetal sex, delivery induction, delivery method (eg, inhalation delivery, cesarean delivery or natural delivery), and neonatal intensive care unit (NICU) needs and prematurity.

The mean concentrations of test variables (referred to herein as biomarkers) were assessed in women with PE compared to controls using nonparametric statistics. Univariate logistic regression model was developed for evaluation biomarkers. At the time of sampling, pregnancy orders were adjusted by logistic regression model. In addition to analyzing the ROC-curve back-off (AUC), biomarkers that show significant differences using the recipient manipulation curve (ROC-curve) were further evaluated by calculating the detection rate at different false positive levels. Parallel analysis was performed on each of the tested biomarkers as well as their different combinations. Further, a subgroup analysis of the ductility with PE, i.e., early and late onset PE, was performed in comparison to the control. A univariate logistic regression model was also used to further calculate fetal outcome (i. E. Admission and early labor and intrauterine growth restriction (IUGR) for NICU) and delivery mode.

Correlation analysis

Correlation analysis (Pearson correlation coefficient) between biomarker, dilator - and systolic blood pressure was performed. The p-value of p? 0.05 was considered significant in all tests.

Correlation between Hpx activity and Hpx concentration was calculated using non - parametric Kendall correlation coefficient. Further, a correlation between Hpx activity and maternal blood pressure (expressed as the highest measured blood pressure within 24 hours prior to delivery) analysis was performed.

A correlation analysis between cell-free Hb (HbF and total Hb), heme, HO-1 and hemopexin concentration was also performed. Furthermore, both heme and HO-1 correlated with both systolic and diastolic blood pressures.

Logistic regression analysis

The detection rate was determined by ROC-curve analysis of each potential biomarker Hpx, HO-1 and heme. 10% and 20% false positive rate. Combined detection of biomarkers was obtained by stepwise logistic regression analysis of biomarkers and ROC-curve analysis.

result

Patient Features

The characteristics of the included patients are shown in Tables 1 and 2. There was a significant difference between the women diagnosed with PE for age, blood pressure, proteinuria, equivalence, gestational age at delivery, and birth weight at the time of sampling and noncomplicated pregnancy (represented as control). Further, significant differences were observed for fetal outcomes (eg, admission and premature birth to NICU) as well as parameters related to maternal outcomes (eg, delivery methods including induction and instrumental delivery). Significant differences were observed in the 10 minute APGAR score between the control and the initial onset PE (but not on late onset PE). There was no significant difference between the groups regarding BMI and fetal sex.

Acellular cell Hb

The concentrations of cell-free HbF, Hp-HbF and Hb- were analyzed in all plasma samples from women with PE and controls (Table 3). A 4-fold increase in HbF concentration was seen in PE patients compared to the control (p-value 0.01). When the PE group was divided into early and late-onset PE, an almost five-fold increase in HbF concentration in the initial onset PE group was observed (p-value 0.006) compared to the control. In the late-onset PE group, a nearly 4-fold increase was observed compared to the control, which is not statistically significant (p-value 0.17).

A statistically significant increase in mean Hp-HbF concentration was observed for women with PE compared to the control (p-value 0.018). A clear trend for the increase could be seen in the initial PE group, but no such difference was found (p-value 0.15).

No significant differences in Hb-total concentrations between PE vs. control (p-value 0.53) or between initial (p-value 0.80) and late onset PE (p-value 0.73) versus control were observed.

Hp and CD163

Analysis of Hp concentration in plasma indicated that increased HbF concentrations were achieved by lower Hp concentrations in PE patients (Table 3). The results showed a highly significant reduction in Hp concentration (p-value < 0.0001) in plasma samples of women with PE compared to the control. In addition, late-onset PE showed a significant decrease (p-value 0.001) compared to the control. In contrast, initial onset PE showed a slight but statistically significant increase in Hp concentration (p-value 0.067) compared to the control.

The Hp-Hb in macrophage receptors to mediate the removal of the soluble complex, shedding (shedded) CD163 in plasma were analyzed ^61-63. Analysis showed a small but not significant (p-value 0.37) increase in the PE group compared to the control group (Table 3). A small but statistically insignificant increase was observed in the late-onset PE group (p-value 0.07 versus control) when the PE group was divided by the early and late-onset PE groups, while a small but statistically significant (P-value 0.35 vs. control).

Hpx

Analysis of the intravascular hem-scavenger protein Hpx showed a highly significant reduction in plasma Hpx concentration in women with PE compared to the control (p-value <0.0001) (Table 3). Dividing the PE groups showed a significant decrease in the initial (p-value <0.0001) and late-onset PE (p-value <0.0001) PE groups compared to the control.

Blood samples were also analyzed for Hpx activity. Plasma Hpx activity was measured in an EDTA plasma sample using an Hpx-MCA substrate (synthesized by Pepscan of Holland Lelystat). Plasma samples (40 μl) were diluted 1: 4 with a substrate solution (0.2 M Tris + 0.9% NaCl pH 7.6 (substrate concentration 80 μM / L)) at a final volume of 200 μl at 37 ° C. Release was measured at 460 nm on a Varioskan spectrophotometer (Thermo Fisher) after a 30 minute incubation (37 C).

Hpx activity was measured spectrophotometrically at the following time points: 0 minutes, 30 minutes (Hpx30), 60 minutes (Hpx60) and 24 hours. The measured values showed the total amount of serine sliced by Hpx at a given time point. If the value was less than 5 after 24 hours, the activity was considered to be extremely low and the sample was excluded from further analysis due to possible damage to the sample. The area under the curve based on Hpx30 and Hpx60 was calculated.

Hpx activity

Eleven samples (8 controls and 3 PEs) exhibited "extremely low values" 24 hours after incubation and were thus excluded from the analysis.

Hpx activity was significantly lower in the PE group than in the control group after 30 min (p = 0.02), 60 min (p = 0.05) and HpxAUC (p = 0.02) (Table 2). However, when the PE patients were divided into early- and late-onset PE, the initial-onset group Hpx30 was evident at 0.81 and was identical to the control (Hpx30 = 0.80) (Table 4). In contrast, the late onset group exhibited a much more significant reduction in Hpx activity than PE on all Hpx activities (Hpx30 = 0.54), generally in general (Table 4).

Interestingly, the ratio between Hpx concentration and Hpx activity was used to assess the risk of progression of early or late onset PE. As can be seen from the table above, the ratio to normal limb was 1.16, while it was 0.85 for the initial onset of PE and 1.28 for the late onset of PE. Thus, if the ratio is lower than that of the control, i. E. Less than 1, there is an increased risk that the initial onset PE will progress whereas a higher than 1.2 ratio will increase the risk of late onset PE progression and Hpx activity will be referred to herein as Hpx30 .

On Hpx  Results for.

Correlation analysis.

Hpx activity was not correlated with Hpx plasma concentration (p = 0.74 for Hpx30). This was not the case in the early initiation group (p = 0.17) or in the late initiation PE group (p = 0.24).

Hpx30 was significantly correlated with diastolic blood pressure in all patients (p = 0.04), and there was a clear trend for correlation between Hpx60 (p = 0.1) and HpxAUC (p = 0.06) (Table 4). When the initial-onset patient was excluded from the analysis, there was a clear correlation between diastolic blood pressure and the respective Hpx30, Hpx60, and HpxAUC (Table 5, FIG. 8). Furthermore, there was a clear trend for correlation between systolic blood pressure and Hpx30 (p = 0.07), Hpx60 (p = 0.17) and HpxAUC (p = 0.11) (Table 5).

Results for blood pressure:

Consistent with previous findings, the inventors have found reduced Hpx activity in patients exhibiting PE. However, the inventors have found that Hpx activity is reduced in patients with only late-onset PE, but not early-onset PE. In contrast and as described herein, Hpx protein concentrations were found to be statistically significantly reduced in both early and late onset PE. Correlation analysis showed a statistically significant inverse correlation between Hpx30 and diastolic blood pressure in all patients, with a similar inverse correlation for Hpx60 and HpxAUC (Table 5). There was a trend toward statistically significant correlation between all Hpx-activity and diastolic blood pressure and a statistically significant correlation with systolic blood pressure when the correlation between just the control and late onset groups were analyzed together.

A1M

Plasma level analysis of heme- and radical scavenger A1M showed a significant increase in plasma A1M concentration (p-value 0.035) in women with PE compared to controls (Table 3). When dividing the PE group, a statistically significant increase was observed in the late-onset PE group (p-value 0.03), but a statistically insignificant increase was seen in the initial onset PE group (p-value 0.26) .

Acellular cell HbF and  Hp correlation

The correlation between plasma HbF and Hp levels was evaluated. Increased plasma acellular HbF concentrations were associated with reduced plasma Hp concentrations (r = -0.335, p-value < 0.0001) when including all patients, controls and women with PE , n = 145) (Figure 1A). Significantly, an increased negative correlation was observed for the PE group (r = -0.437, p-value (FIG. 1B)) when compared to the correlation in the control (FIG. 1B) <0.0001, n = 98) In the control group, a weak positive correlation was observed (r = 0.142, p-value 0.33, n = 47). Similar correlations were observed for Hp versus Hp-HbF and Hp versus Hb-none, none of which reached statistical significance (Hp vs. Hp-HbF r = -0.05, p-value 0.52; Hb-total r = 0.03, p-value 0.73).

Hp Isofom and Acellular cell HbF , Hpx  And A1M's  Correlation Between Levels

We used western blots to identify predominant Hp-isoforms (1-1, 1-2, or 2-2) in patient plasma samples (Figure 2A). As can be seen in Figure 2B, Hp 1-2 (C, 45%; PE, 41%) and 2-2 (C, 43%; PE, A similar distribution of different isoforms was observed in both the control and the PE by the predominant presence of &lt; RTI ID = 0.0 &gt; Repeating PE groups in early and late-onset PE showed similar distribution 1-1 (early, 13%; late, 15%), 1-2 (early, 45%, late, 40% 42%; late, 45%). Further, the relationship between plasma levels of Hp-isoform and cell-free HbF, Hp-HbF, Hb-whole, Hp, CD163, Hpx and A1M was analyzed (FIGS. 2C to 2D). A noticeable increase in acellular HbF concentration was observed in the female Hp 2-2 group with PE (Fig. 2C). A small but similar increase in Hp-HbF concentration was observed in the Hp 2-2 PE group compared to the control (Figure 2D). No additional significant relationship with Hp isoform was observed.

Biomarkers and  Correlation analysis between disease severity

Correlation analysis using Pearson's correlation coefficient showed that both Hpx and blood pressure (P <0.05) were significant in both the systolic (r = -0.511, p-value <0.00001, n = 145) (Fig. 3). The results are shown in Fig. No statistically significant correlation was observed for any other biomarkers and blood pressure.

Diagnostic Tools and Clinical As a predictor Biomarker  evaluation

The logistic regression model was used to evaluate the usefulness of the biomarkers described as diagnostic markers for PE. Comparing women with PE to controls, a significant difference was detected (p-value <0.0001) for HbF, A1M and Hpx, but not for Hp and CD163. Each significantly altered biomarker was able to diagnose PE (adjusted for pregnancy), but Hpx showed a high level of significance and diagnostic sensitivity of 64% with a false positive rate of 5% with AUC 0.87 (Table 6, Fig. 4C) . The combination of Hpx, A1M and HbF was not significant (p-value for HbF 0.08), but with a false positive rate of 5% with a diagnostic yield of 69% with AUC 0.88 (Table 6, Fig. 4A). The combination of Hpx and A1M was significant, with a false-positive rate of 5% and AUC 0.87 with a diagnosis of 66% (Table 6, Fig. 4B).

Prediction of fetal and maternal outcomes

In addition to biomarker testing to support the diagnosis of PE, the present inventors have tested whether biomarkers can predict various fetal and maternal outcomes. This was done using a logistic regression model similar to the PE model. Tested fetal outcomes are hospitalization, IUGR and premature birth for NICU. The tested maternal outcomes are induction labor, cesarean section and inhalation. The biomarkers HbF (p-value 0.001), Hpx (p-value 0.008) and Hp (p-value 0.03) each had potential as a predictive biomarker for "hospitalization for NICU". However, in a combined logistic regression model, they proved to be insignificant. The biomarkers Hpx (p-value 0.0003, AUC = 0.71) and CD163 (p-value 0.03, AUC = 0.61) showed potential as biomarkers for prematurity. In combination, these two biomarkers demonstrated a slightly more significant association with preterm birth (p-value 0.001 and p-value 0.025, AUC 0.72).

None of the biomarkers showed any predictive value for induction or inhalation. Hpx was significantly associated with cesarean section (p-value 0.009, AUC 0.62).

Study II - Sampling during 6 to 20 weeks of gestation

Patients and samples

The study was approved by the Ethics Committee of St Georges University Hospital in London, UK. All participants signed a preliminary agreement before inclusion. Women who attend routine prenatal care are women in London. He visited Georges Hospital Obstetrics and Gynecology Department and was hired in 2006 and 2007.

The length of the gestation period was calculated from the last menstrual period, and then confirmed by measuring the ultrasonic head hips length. Maternal venous blood samples were collected at 6 to 20 weeks gestation (mean 13.7) in 5 ml vacuum blood vessels (Becton Dickinson, Franklin Lakes, NJ) without additives. After coagulation, the samples were centrifuged at 2000 xg for 10 minutes at room temperature, then the serum was separated and stored at-80 C until further analysis.

All pregnancy outcomes - data were obtained from the main delivery database and verified for each individual patient. PE was expressed as in Study I of this specification.

As in Study I, a normal pregnancy was defined as a pregnancy with a normal blood pressure of 37 + 0 weeks or later. Noncompliant pregnancy (control) samples were adopted as successive cases for the same time period.

gun Hb , HbF , A1M , Hp and Hpx's  Measure

HbF-concentrations in serum samples (i.e., cell-free HbF) were measured by sandwich ELISA using polyclonal antibodies as described in Study I. The A1M concentration was determined by radioimmunoassay as described in Study I. Hb-total, Hp and Hpx concentrations were serum samples using an ELISA quantitation kit for each marker as described in Study I.

Statistical analysis

SPSS statistical version 21.0 for Apple computers was used with statistical software R studio version (0.98.1062). p value ≤ 0.05 was considered significant in all analyzes. Significant differences between the groups for the biomarkers HbF, Hb-whole, Hp, Hpx and A1M were calculated by the one-way ANOVA. Due to the difference in pregnancy command when performing the Doppler ultrasound in PE and the control group, UtAD is given multiple of the median value according to the average value by the literature [Velauthar et al] (Multiples of the Median: MoM) - it was converted to value ^64.

Staged regression analysis is a commonly used method for ongoing predictive models, but has been criticized ⁶⁵ . Therefore, the inventors have also attempted to demonstrate the results by developing two or more recently developed statistical methods, Lasso regression and boosted tree regression, These methods were compared. The method was compared by ROC-curve area. To prove the prediction results, the data sets were randomized into training cohorts (2/3) and used to develop the test cohorts (1/3) used to test the prediction models and their predictive capabilities.

The final model of the biomarker and maternal characteristics was constructed on the backward stepwise logistic regression. Separate analyzes were performed for early initiation PE and late initiation PE. All regression parameters and parameters combined with the ROC curve were performed and the predicted rates at different false positive rates (FPR) were calculated. The optimal prediction rate / FPR is shown as the point in the ROC-curve closest to the upper left corner.

result

Demography

In total, 520 women were included, 86 of whom had PE (case), 65 of which were spontaneous preterm birth (SPTB), 7 of whom had IUGR complications, 10 of whom were pregnant with hypertension ), One patient had IUGR and placenta abruption, three had separate placental abruption (no PE or IUGR), and 2 had essential hypertension. A total of 347 women (37 weeks gestational period) who had no complications and who had term pregnancies were included as controls.

Matrix characteristics are shown in Table 8. Among the 86 women with PE, 28 were delivered 37 + 0 weeks before gestation. Of these, 17 delivered 34 + 0 weeks before gestation with significantly lower birth weight compared to the control group. Essential hypertension groups without PE (n = 2) and exfoliation (n = 3) were excluded after analysis because of their small size.

There was no statistically significant difference in serum sampling time between case and control group.

Biomarker

Table 9 shows the serum levels of the biomarkers HbF, Hp, A1M, Hb-whole, Hp and Hpx.

The mean concentration of HbF (10.8 ㎍ / ㎖, p = 0.02) in the PE group was significantly higher in the control group (5.6 / / ml). HbF is the total HbF as compared to the HbF of mainly non complications described in Study I. The mean A1M concentration was also significantly increased (17.3 μg / ml versus 15.5 μg / ml, p = 0.03). The mean Hpx concentration in the PE group was significantly lower (p = 0.05), compared with 1143 ㎍ / ㎖ in the control group, to 1062 ㎍ / ㎖. There was a trend towards a slightly higher Hp concentration (1102 mg / ml) in the PE group compared to the control (971 mg / ml) but was not significant (p = 0.089). PIH or IUGR was similar to that of the control group (data not shown). The SPTB group presented significantly lower levels of Hb-total (201 μg / ml vs. 297 μg / ml, p = 0.05) and Hpx (1061 μg / ml vs 1143, p = 0.05). UtAD MoM values were significantly higher in the PE group than in the control group (1.18 vs. 0.95 p <0.0001).

Logistic regression analysis

The biomarker's ability to predict PE was tested in a logistic regression model. A corresponding ROC-curve was generated to visualize the predicted values. All biomarkers were tested individually as well as in combination to find the best predictive value. Significant results are outlined in Table 10, and the ROC-curve is shown in FIG.

Subsequently, despite the significantly increased serum HbF concentration in PE - progressed patients, they showed limited predicted values when used alone (FPR 10% to PR 15%). A1M showed similar predictions (FPR 10% to PR 19%). Hpx showed the best individual predictability for PE (FPR 10% to PR 42%). A1M, HbF, and Hpx (PR 62% in FPR 10).

To compare PE and control, all measurements of maternal characteristics were tested alone and in combination using logistic regression analysis.

The combination of maternal characteristics (equivalence, diabetes, pre-pregnancy hypertension) and biomarkers (HbF, A1M and Hpx) increased PR to 62% at FPR 10% (Table 10, Figure 7) Showed a similar prediction rate (PR 57% at 10% FPR)

Early early late onset preeclampsia

We found elevated levels of HbF in both early and late-onset PE groups (Table 11).

A1M levels were significantly higher only in the late-onset group (p = 0.01) (Table 11). Hpx protein concentrations were lower in both groups, but only in the initial onset PE group (p = 0.04) (Table 11). UtAD PI MoM was significantly elevated in the early onset group (1.63 vs. 0.95, p <0.00001) but only slightly in the late onset group, and this difference was not statistically significant (1.06 vs. 0.95, p = 0.06). There was no significant difference in Hb-total or Hp in one of the study groups.

The logistic regression model for early- and late-onset PEs for the biomarkers tested showed predictability for FPR 10% to HbF 23% (but only in late-onset PE groups). A1M was statistically significant only in the late onset group (p = 0.01), Hpx was statistically significant only for the initial onset group, and PR 32% in FPR 10%.

UtAD performed best in the early onset group with FPR 10% to PR 57%, but statistically significant, even in the late onset group.

None of the biomarkers were statistically significant in combination with maternal characteristics or UtAD in either the early or late initiation group.

Argument

The aim of this study is to demonstrate previous findings that serum levels of cell-free HbF and A1M are already elevated in the first 3 months of pregnancy and that they are useful as predictive first 3 months biomarkers during subsequent development of PE. In this study, the cohort size is larger and better reflects the normal incidence of PE. In addition, the study also assesses the effects of biologically relevant heme- and Hb-scavenging proteins Hp and Hpx.

The main findings in this paper confirm that both HbF and A1M are subsequently elevated significantly in sera from pregnant women who subsequently developed PE (Table 9). Increased serum levels of HbF are likely to be caused by defective placental hematopoiesis, reflecting placental oxidative stress. The data indicate that HbF and A1M have the potential as biomarkers for the first 3 months and the first 2 months of the predicted for PE. Further, the heme scavenger Hpx also exhibits a good predictive value and is therefore also presented as an additional potential biomarker for PE. The UtAD index showed a higher PI MoM value mainly in the initial onset group. This is in full agreement with previously published results from several study groups. The higher PI in the early initiation group reflects increased resistance in the uterine artery as a result of shallow involvement of the maternal decidua spiral arteries (characteristic of early onset PE, but less common in late onset PE).

Interestingly, the data showed that cell-free Hb-total and Hpx were significantly lower in patients who were preterm. The low enzyme activity of Hpx is known to weaken endothelial inflammation. Thus lower levels of Hpx could be attributed to increased maternal inflammation seen in both PE and prematurity. Future studies need to read more carefully the role of Hpx in preterm labor.

The specific first three months of screening for adverse pregnancy outcomes is important because it provides clinicians with tools to target and personalize patient surveillance rather than subsequent general screening programs in pregnancy. By confirming high-risk pregnancies, cognitive strategies and prophylactic treatment can be initiated. To date, prophylactic treatment is low-dose acetylsalicylic acid (ASA). There is a significant risk reduction (RR = 0.47) especially for early onset and severe PE if treatment is initiated 16 weeks prior to gestation. The number needed to treat (NNT) may be as low as 7 to prevent severe PE in a confirmed high-risk pregnancy. The use of ASA is inexpensive and has few side effects given the recommended low dose (75 mg). Prophylactic treatment should be initiated at the end of the first three months in order to have optimal effect. Taking this into account, if it is possible to predict PE at the end of the first 3 months or at the beginning of the second 3 months, this is possible and possibly combined with other established screening programs for Down syndrome.

conclusion:

HbF, A1M, and Hpx, measured at maternal serum at the end of the first 3 months of pregnancy and at the start of the second 3 months, are potential predictive biomarkers for the postinflammation of PE. The three proteins are physiologically relevant because increased amounts of acellular HbF have been described to be involved in pathology and potentially consuming physiological hem-scavenging proteins. Furthermore, the predictive power of the three biomarkers is increased by combination with uterine artery Doppler ultrasound and / or maternal features.

references

                         SEQUENCE LISTING <110> A1M PHARMA AB   <120> Biomarkers for preeclampsia <130> WO / 2016/146647 <140> PCT / EP2016 / 055619 <141> 2016-03-16 <150> DK PA 2015 70146 <151> 2015-03-16 <150> DK PA 2015 70794 <151> 2015-12-03 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 183 <212> PRT <213> Homo sapiens <400> 1 Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Gln Val Gln Glu Asn Phe 1 5 10 15 Asn Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala Ile Gly Ser             20 25 30 Thr Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr Val Ser Thr         35 40 45 Leu Val Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser Met Thr Ser     50 55 60 Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly Ala Tyr Glu 65 70 75 80 Lys Thr Asp Thr Asp Gly Lys Phe Leu Tyr His Lys Ser Lys Trp Asn                 85 90 95 Ile Thr Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp Glu Tyr Ala             100 105 110 Ile Phe Leu Thr Lys Lys Phe Ser Arg His His Gly Pro Thr Ile Thr         115 120 125 Ala Lys Leu Tyr Gly Arg Ala Pro Gln Leu Arg Glu Thr Leu Leu Gln     130 135 140 Asp Phe Arg Val Val Ala Gln Gly Val Gly Ile Pro Glu Asp Ser Ile 145 150 155 160 Phe Thr Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu Gln Glu Pro                 165 170 175 Glu Pro Ile Leu Ile Pro Arg             180 <210> 2 <211> 201 <212> PRT <213> Homo sapiens <400> 2 Met His His His His His His His Gly Gly Gly Gly Gly Gly Ile Glu 1 5 10 15 Gly Arg Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Gln Val Gln Glu             20 25 30 Asn Phe Asn Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala Ile         35 40 45 Gly Ser Thr Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr Val     50 55 60 Ser Thr Leu Val Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser Met 65 70 75 80 Thr Ser Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly Ala                 85 90 95 Tyr Glu Lys Thr Asp Thr Asp Gly Lys Phe Leu Tyr His Lys Ser Lys             100 105 110 Trp Asn Ile Thr Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp Glu         115 120 125 Tyr Ala Ile Phe Leu Thr Lys Lys Phe Ser Arg His His Gly Pro Thr     130 135 140 Ile Thr Ala Lys Leu Tyr Gly Arg Ala Pro Gln Leu Arg Glu Thr Leu 145 150 155 160 Leu Gln Asp Phe Arg Val Val Ala Gln Gly Val Gly Ile Pro Glu Asp                 165 170 175 Ser Ile Phe Thr Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu Gln             180 185 190 Glu Pro Glu Pro Ile Leu Ile Pro Arg         195 200 <210> 3 <211> 549 <212> DNA <213> Homo sapiens <400> 3 ggccctgtgc caacgccgcc cgacaacatc caagtgcagg aaaacttcaa tatctctcgg 60 atctatggga agtggtacaa cctggccatc ggttccacct gcccctggct gaagaagatc 120 atggacagga tgacagtgag cacgctggtg ctgggagagg gcgctacaga ggcggagatc 180 agcatgacca gcactcgttg gcggaaaggt gtctgtgagg agacgtctgg agcttatgag 240 aaaacagata ctgatgggag gtttctctat cacaaatcca aatggaacat aaccatggag 300 tcctatgtgg tccacaccac ctatgatgag tatgccattt ttctgaccaa gaaattcagc 360 cgccatcatg gacccaccat tactgccaag ctctacgggc gggcgccgca gctgagggaa 420 actctcctgc aggacttcag agtggttgcc cagggtgtgg gcatccctga ggactccatc 480 ttcaccatgg ctgaccgagg tgaatgtgtc cctggggagc aggaaccaga gcccatctta 540 atcccgaga 549 <210> 4 <211> 603 <212> DNA <213> Homo sapiens <400> 4 atgcatcacc atcaccatca ccatcacggt ggaggagggg gtatcgaggg ccgcggccct 60 gtgccaacgc cgcccgacaa catccaagtg caggaaaact tcaatatctc tcggatctat 120 gggaagtggt acaacctggc catcggttcc acctgcccct ggctgaagaa gatcatggac 180 aggatgacag tgagcacgct ggtgctggga gagggcgcta cagaggcgga gatcagcatg 240 accagcactc gttggcggaa aggtgtctgt gaggagacgt ctggagctta tgagaaaaca 300 gatactgatg ggaggtttct ctatcacaaa tccaaatgga acataaccat ggagtcctat 360 gtggtccaca ccacctatga tgagtatgcc atttttctga ccaagaaatt cagccgccat 420 catggaccca ccattactgc caagctctac gggcgggcgc cgcagctgag ggaaactctc 480 ctgcaggact tcagagtggt tgcccagggt gtgggcatcc ctgaggactc catcttcacc 540 atggctgacc gaggtgaatg tgtccctggg gagcaggaac cagagcccat cttaatcccg 600 aga 603

Claims (32)

A biomarker panel comprising a biomarker for preeclampsia, the biomarker comprising:
i) hemopexin (Hpx) and alpha-1-microglobulin (A1M),
ii) a biomarker panel comprising a biomarker for preeclampsia, further comprising one or more biomarkers selected from Hpx, A1M and free HbF, optionally selected from Hp-HbF, Hp, HO-1, The biomarker panel according to claim 1, comprising a biomarker for preeclampsia, comprising HO-1 and / or heme as biomarker (s) for preeclampsia. 3. The biomarker panel according to claim 1 or 2, further comprising a biomarker for preeclampsia, further comprising Hp-HbF and / or haptoglobin as biomarker (s) for preeclampsia. 4. The biomarker panel according to any one of claims 1 to 3, comprising a biomarker for preeclampsia for use in predicting or diagnosing preeclampsia or for evaluating the risk of progression of preeclampsia. 5. The biomarker of claim 4, for use as a marker for early or late onset of preeclampsia. 6. The method according to any one of claims 1 to 5, wherein the marker (s) is / are selected from the group consisting of a bioassay for preeclampsia, which is measured in blood, plasma, serum, CSF, urine, placental biopsy, uterine fluid, A biomarker panel comprising a marker. 7. The method of claim 6 wherein the level of HO-1 and / or haptoglobin in the plasma sample from the pregnant female is at least 1.1 times less than the reference value, and the level of alpha-1-microglobulin, If appropriate, the level of free non-protein-bound fetal hemoglobin and the level of heme is at least 1.1 times greater than the reference value, said reference value being at least one of a risk of not suffering from pre-eclampsia in the same gestational age as said test sample, If the pregnancy females are determined to be in pregnancy or if the reference value is adjusted to correlate with the gestational age of the test sample, then the pregnant females may have preeclampsia, or a biomarker for preeclampsia at an increased risk of progression of preeclampsia Biomarker panel. 8. The biomarker panel according to any one of claims 1 to 7, wherein said sample comprises a biomarker for preeclampsia taken from said pregnant female at 6 to 20 weeks of gestational age. 9. The method according to any one of claims 6 to 8, wherein the hemopexin level in the plasma sample from the pregnant female taken at 6 to 20 weeks gestational age is 1.0 mg / ml or less, the alpha-1-microglobulin level Is greater than or equal to 15.5 μg / ml, and where appropriate, the level of free total fetal hemoglobin is greater than or equal to 5.6 ng / ml, the pregnant female may have a pre-eclampsia biomarker Includes biomarker panels. 10. A method according to any one of claims 3 to 9, wherein the Hp-HbF level in the plasma sample from said pregnant female taken at 6 to 20 weeks of gestational age is greater than or equal to 0.7 g / ml and the level of HO- wherein said pregnant females have preeclampsia or are at increased risk of progression of preeclampsia if said fetus is less than or equal to ng / ml and / or the level of heme is greater than or equal to 60 μM. 8. The biomarker panel according to any one of claims 1 to 7, wherein said sample comprises a biomarker for preeclampsia taken from said pregnant woman at 34 to 40 weeks of gestational age. 12. The method according to claim 11, wherein the level of hemopexin in plasma samples from pregnant females taken at 34-40 weeks gestational age is below 0.85 mg / ml, the level of alpha-1-microglobulin is above 30 g / The biomarker panel comprising a biomarker for preeclampsia wherein said pregnant females have preeclampsia or are at increased risk of progression of preeclampsia if the level of free non-protein coupled fetal hemoglobin is greater than 6.5 ng / ml. The method according to claim 11 or 12, wherein the Hp-HbF and HO-1 are included as biomarkers and the Hp-HbF level in plasma samples from the pregnant females taken at 34-40 weeks gestational age is less than 0.5 & Or less, the level of free non-protein-coupled fetal HO-1 is less than 5.0 ng / ml, and the level of heme is greater than 68 μM, the pregnant female has preeclampsia or preeclampsia A biomarker panel including a biomarker for the biomarker. For the diagnosis of preeclampsia or for the diagnosis of preeclampsia, including the following steps to determine whether a pregnant woman has or does not have preeclampsia, or whether it is in an increased risk of progression of preeclampsia or in an increased risk How to Assist:
(a) obtaining a biological sample from a pregnant woman;
(b) measuring biomarker levels selected from biomarker panels i) and ii), wherein i) is Hpx and A1M; ii) measuring the biomarker level, wherein i) or ii) optionally comprises at least one of the following biomarkers Hp-HbF, Hp, HO-1, heme: Hpx, A1M and free HbF , And
(c) comparing the level of the biomarker (s) measured in the sample with a reference value. A method for monitoring the progression or regression of pre-eclampsia comprising the steps of:
(a) measuring a biomarker level selected from biomarker panels i) and ii) in a first biological sample isolated from a pregnant female mammal, such as a blood, urine or plasma sample, wherein i) is Hpx and A1M ; ii) measuring the biomarker level, wherein i) or ii) optionally further comprises at least one of the following biomarkers Hp-HbF, Hp, HO-1, heme: Hpx, A1M and free HbF ;
measuring the level of said biomarker selected under (a) in a second biological sample isolated from said pregnant female mammal, such as a blood, urine, serum or plasma sample, at a time after step (b); and
(c) comparing the measured values in steps (a) and (b)
i) an A1M level in the second sample and, if appropriate, an increase relative to the level (s) in the first sample of HbF, Hp-HbF, heme, and /
ii) the decrease in Hpx level in the second sample and, if appropriate, the level (s) in the first sample of Hp, HO-1 level (s)
Indicates progress of preeclampsia; Wherein the decrease in i) and / or the increase in ii) described above is indicative of pre-eclampsia regression. 16. The method according to claim 14 or 15, wherein Hp is included as a biomarker. 17. The method according to any one of claims 14 to 16, wherein the combination of biomarkers further comprises free HbF. 18. The method according to any one of claims 14 to 17, wherein the sample (in claim 14) or the first sample (in claim 14) is taken at least 6 weeks to the gestational age. 19. The method of claim 18, wherein the sample is taken at 6 to 20 weeks or at 12 to 14 weeks. 19. The method of claim 18, wherein the sample is taken at 34 to 40 weeks. Hemoglobin, haptoglobin and / or free non-protein coupled fetal hemoglobin as predictive markers (s) for delivery of the baby by premature and / or caesarean section. Fetus for hospitalization for NICU, hemopexin as predictive marker (s), haptoglobin and / or free non-protein coupled fetal hemoglobin. Hemopexcin for use as a marker or predictive marker for preeclampsia. To assist in the diagnosis or diagnosis of pre-eclampsia, including the following steps to determine whether the pregnant female has or does not have pre-eclampsia or whether it is in an increased risk of progression of preeclampsia or in an increased risk How to:
(a) obtaining a biological sample from a pregnant woman;
(b) measuring Hpx levels in the sample; And
(c) comparing the Hpx level in the sample to a reference value. 26. The method of claim 24, wherein the sample is taken from the pregnant woman at 6 to 20 weeks of gestational age to assist in the diagnosis or diagnosis of preeclampsia. For the purpose of diagnosing pre-eclampsia, or for assisting diagnosis, to determine whether a pregnant female has or does not have preeclampsia, or whether it is in an increased risk or at risk of progression of preeclampsia Way:
(a) obtaining a biological sample from a pregnant woman in a pregnancy order of 6 to 20 weeks;
(b) measuring the level of Hpx and optionally the level of biomarker (s) total HbF and / or A1M; And
(c) comparing the measured biomarker (s) level in the sample to a reference value. CLAIMS 1. A method for monitoring progression or regression of preeclampsia comprising the steps of:
(a) the level of Hpx and optionally the level of said biomarker (s) total HbF and / or A1M in a first biological sample, such as a blood, urine or plasma sample isolated from a female mammal pregnant at 6 to 20 weeks gestational age Measuring a level;
(s) total HbF and / or A1M levels in a second biological sample, such as a blood, urine or plasma sample isolated from said pregnant female mammal at a later time (b) Measuring; And
(c) comparing the measured values in steps (a) and (b)
i) an increase in the total HbF and / or A1M level (s) in the second sample relative to the total HbF and / or A1M level (s) in the first sample, and /
ii) the reduction of the Hpx level (s) in the second sample relative to the Hpx level (s) in the first sample
Indicates progression of preeclampsia; Wherein the decrease in i) and / or the increase in ii) described above is indicative of pre-eclampsia regression. 28. The method according to any one of claims 23 to 27, wherein Hpx is used as a marker with HO-1. A method for determining whether a pregnant female has or does not have preeclampsia, or whether the preeclampsia is in an increased risk of progression or not in an increased risk of progression of preeclampsia, comprising the steps of:
(a) obtaining a biological sample from a pregnant woman;
(b) measuring the A1M level in the sample; And
(c) comparing the A1M level in the sample to a reference value. 30. The method of claim 29, wherein said sample is taken from a pregnancy at 6 to 20 weeks or 34 to 40 weeks of gestation. For the diagnosis of preeclampsia or for the diagnosis of preeclampsia, including the following steps to determine whether the pregnant female has or does not have preeclampsia, or whether it is in an increased risk of progression of the preeclampsia or in an increased risk Method for:
(a) obtaining a biological sample from a pregnant woman in a pregnancy order of 6 to 20 weeks;
(b) measuring the level of A1M and optionally the level of biomarker (s) total HbF and / or Hpx; And
(c) comparing the measured biomarker (s) level in the sample with a reference value. CLAIMS 1. A method for monitoring the progression or regression of preeclampsia,
(a) the level of A1M in a first biological sample, such as a blood, urine or plasma sample isolated from a female mammal gestating at 6 to 20 weeks or 34 to 40 weeks of gestational age, and optionally the biomarker (s) total HbF and / Or the level of Hpx;
(b) measuring the level of A1M and optionally the level of biomarker (s) total HbF and / or Hpx in a second biological sample such as a blood, urine or plasma sample isolated from said pregnant female mammal at a later time step; And
(c) comparing the measured values in steps (a) and (b)
i) an increase in the total HbF and / or A1M level (s) in the second sample relative to the total HbF and / or A1M level (s) in the first sample, and /
ii) the decrease in Hpx level (s) in the second sample relative to the Hpx level (s) in the first sample is
Indicates progression of preeclampsia; Wherein said decreasing of i) and / or the increase of ii) described above is indicative of preeclampsia regression.

Images